Design of Trehalose-Based Amide/Sulfonamide C-type Lectin Receptor Signaling Compounds

Article abstract of DOI:10.1002/cmdc.202000775

Mincle agonists have been shown to induce inflammatory cytokine production, such as tumor necrosis factor-alpha (TNF) and promote the development of a Th1/Th17 immune response that might be crucial to development of effective vaccination against pathogens

Full text of DOI:10.1002/cmdc.202000775

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ChemMedChem
Design of Trehalose-Based Amide/Sulfonamide C-type
Lectin Receptor Signaling Compounds
Omer K. Rasheed,[a, e] Cassandra Buhl,[b] Jay T. Evans,[b, c] and Kendal T. Ryter*[a, c, d]
development of new, safe, and effective vaccine is still required
for emerging new pathogens, re-emerging pathogens, and the
inadequate protection conferred by some existing vaccines.[1]
Newly arising immunization approaches such as subunit
vaccines have increased the need for potent and selective
adjuvants, particularly adjuvants inducing cell-mediated im-
mune responses.[2] A breakthrough was the identification of the
macrophage inducible C-type lectin (Mincle, Clec4e, or Clecf9)
as a pattern recognition receptor (PRR) on the surface of
macrophages and dendritic cells activated through ligation of
trehalose dimycolate (TDM), the major component of Mycobac-
terium cell wall and its synthetic analogues trehalose-6,6’-
dicorynomycolate (TDCM) and trehalose dibehenate (TDB; Fig-
ure 1).[3,4] TDM and TDB have the ability to stimulate the innate
and early adaptive immune response that has led to an interest
in the potential of these molecules as adjuvants.[5] In particular,
TDB has shown promise as a vaccine adjuvant for both
tuberculosis and HIV when formulated in the CAF01 liposome
system.[6] TDM, TDB, and TDCM,[7a] demonstrated that these
glycolipids are potent agonists,[7] although TDM and TDCM
have proven to be too reactogenic for human vaccine
applications.[8] The development of TDB and other trehalose
diesters (TDEs),[9–11] has partially met the need for new adjuvants
although the physiochemical properties of these adjuvants,
being very lipophilic with limited aqueous solubility, limits their
application to complex liposomal formulations.[12] Brartemicin, a
trehalose-based natural product, has been reported as a high-
affinity ligand of the carbohydrate recognition domain (CRD) of
the C-type lectin Mincle.[13] In recent years several lipidated
diaryl derivatives have been described to elicit Th17 cytokines
in a Mincle-dependent fashion with improved adjuvant activity
and physical properties over TDB.[10b,14] The synthesis and
Mincle agonists have been shown to induce inflammatory
cytokine production, such as tumor necrosis factor-alpha (TNF)
and promote the development of a Th1/Th17 immune response
that might be crucial to development of effective vaccination
against pathogens such as Mycobacterium tuberculosis. As an
expansion of our previous work, a library of 6,6’-amide and
sulfonamide α,α-d-trehalose compounds with various substitu-
ents on the aromatic ring was synthesized efficiently in good to
excellent yields. These compounds were evaluated for their
ability to activate the human C-type lectin receptor Mincle by
the induction of cytokines from human peripheral blood
mononuclear cells. A preliminary structure–activity relationship
(SAR) of these novel trehalose diamides and sulfonamides
revealed that aryl amide-linked trehalose compounds demon-
strated improved activity and relatively high potency cytokine
production compared to the Mincle ligand trehalose dibehen-
ate adjuvant (TDB) and the natural ligand trehalose dimycolate
(TDM) inducing dose-dependent and human-Mincle-specific
stimulation in a HEK reporter cell line.
Vaccination against infectious pathogens is perhaps the most
promising and potentially effective tool in combating global
morbidity/mortality and health disparity from infectious dis-
eases. Notwithstanding the great success of vaccines, the
[a] Dr. O. K. Rasheed, Dr. K. T. Ryter
Department of Chemistry and Biochemistry,
University of Montana
32 Campus Drive, Missoula,
MT 59812 (USA)
E-mail: Kendal.ryter@mso.umt.edu
[b] C. Buhl, Dr. J. T. Evans
Department of Biomedical and Pharmaceutical Sciences
University of Montana
32 Campus Drive, Missoula,
MT 59812 (USA)
[c] Dr. J. T. Evans, Dr. K. T. Ryter
Center for Translational Medicine,
University of Montana
32 Campus Drive, Missoula,
MT 59812 (USA)
[d] Dr. K. T. Ryter
Center for Biomolecular Structure and Dynamics
University of Montana
32 Campus Dr., Missoula,
MT 59812 (USA)
[e] Dr. O. K. Rasheed
Current Address: Inimmune Corp.
1121 E. Broadway, Suite 121, Missoula,
MT 59802 (USA)
Figure 1. Representative Mincle ligands: trehalose dimycolates (TDM), α-
trehalose diesters (αTDE’s, B16), trehalose dicorynomycolates (TDCM),
trehalose dibehenate (TDB), brartemicin analogues, and UM1024.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000775
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evaluation of the first relatively small 6,6’-aryl ester trehalose
(UM1024; Figure 1) was recently reported, with demonstrated
ability to promote a Th1/Th17 immune response invitro and
invivo.[15] The results reported here further explore the structural
basis for the activity of this new class of Mincle signaling
compounds and identify structural motifs responsible for
immune activation with the potential to improve physiochem-
ical properties, in particular solubility, for use as vaccine
adjuvants. Also, evaluation of Mincle crystal structures[16]
suggested improved calcium metal/ligand affinity in the
proposed binding pocket in addition to improved interactions
of adjacent glutamine and arginine moieties through the
replacement of the 6,6’-ester with amide or sulfonamide
functionality.
As our continued interest in exploring the potential utility
of 6,6’-aryl α,α-d-trehalose analogues on Mincle-binding and
cytokine signaling as vaccine adjuvants have generated several
novel compounds with improved potency and solubility over
the currently available Mincle ligands such as TDB and
TDM.[13,14,17,18] The natural extension of structure–activity studies
is the synthesis of 6,6’-aryl amide α,α-d-trehalose and 6,6’-aryl
sulfonamide α,α-d-trehalose analogues related to previously
prepared ester analogues[17b] to investigate the impact of the
aryl-trehalose linkage on Mincle selective cytokine signaling.
6,6’-diamino trehalose (3) is a key intermediate for the synthesis
of the targeted trehalose diamides (TDA’s) was obtained as
shown in Table 1. Mesylation of primary alcohols of hexa-O-
benzolated trehalose[18a] (1) followed by azidation under
standard conditions furnished the diazide in good yield. In the
catalytic hydrogenation of diazide (2), the presence of
hydrochloride is necessary to block the well-known oxygen-to-
nitrogen benzoyl migration. Alkoxy aryl acids that were not
commercially available were prepared from the corresponding
hydroxy arylbenzoates. Methyl 3,5-dihydroxybenzoate and
methyl 3,4,5-dihydroxybenzoate were alkylated using requisite
alkyl bromides followed by ester hydrolysis providing the
desired benzoic acid derivatives in excellent yields over two
steps.[19] Similarly, the aliphatic branched carboxylic acid (B16)
was synthesized starting from diethyl malonate over 4 steps in
good yield.[8a] The trehalose diamide derivatives were then
assembled by HATU-mediated amidation of 6,6’-diamino treha-
lose (3) with carboxylic acid (2.5 equiv. per equiv. of OBz-
protected trehalose). Having accomplished the synthesis of the
protected diamides (4), all that remained was the global
Table 1. Synthesis of a focused library of 6,6’-diaryl and alkyl amide trehalose Mincle signaling compounds.
Compound
R
Yield [%]
Coupling
product
Compound
R
Yield [%]
Coupling
product
Deprotection
87
Deprotection
78
UM1088
86
5f
85
5a
5b
5c
83
74
80
84
82
86
5g
5h
5i
79
81
76
80
84
78
5d
5e
79
86
82
81
5j
77
82
81
89
5k
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deprotection of benzoyl groups using conventional NaOMe in
MeOH conditions to obtain the target compounds (UM1088,
5a–k; Table 1) that were fully characterized by spectroscopic
techniques (see the Supporting Information for details).
for the activity of TDM or TDB. We presume this difference is
due to conformation obtained in the drying process of the
compound which is critical for the activity/activation of these
PRR’s. Studies are ongoing to further explore the tertiary
structural requirements of Mincle receptor binding and activa-
tion by what we presume to be nanoparticles.
Initially, we investigated the ability of aryl amide analogue
UM1088 for the production of IL-6 in comparison with our
previously reported potent relatively small Mincle signaling
compound UM1024 (Figures 1 and 2A).[17b] UM1088 was
observed to be equipotent to UM1024 and surprisingly did not
exhibit a decline in potency at high concentrations as observed
for UM1024 and most other Mincle ligands tested to date.
Currently, we are postulating that compound aggregation at
high concentrations is responsible for this observation as we do
not observe activation-induced cell death at high compound
concentrations of UM1024 and we observe a similar profile for
compounds such as 5h.
Analysis of the remaining library of compounds containing
amide functionality was tested for IL-6 production with Mincle
signaling compounds, TDB, TDM, and UM1088 as positive
controls (Figure 2B). Compounds containing the sterically bulky
group, that is, tert-butyl group, at positions 3 and 5 (5k) led to
significant production of IL-6 as compared to less sterically
hindered groups at 3 and 5 positions. For example, 5d or ethyl
at 3 and 5 position (5e) resulted in a loss of activity. In the
same way, 3,5 diether derivatives with the extension of the
Mincle agonists have been shown to induce inflammatory
cytokine production, such as tumor necrosis factor-alpha (TNF),
and promote the development of a Th1/Th17 immune response
that is associated with and dependent on induction of cytokines
such as IL-6, IL-1β, and IL-23.[20] Therefore, for preliminary
screening, the synthesized compounds were assayed for IL-6,
TNF, and IL-1β production in human PBMC’s (Figure 2).[8a,21]
Briefly, compounds were serially diluted in ethanol, applied to
tissue culture plates and the solvent was evaporated. Freshly
isolated human PBMCs were added and incubated with the
compound for 18–24 hours. Supernatants were collected and
evaluated for IL-6 levels by ELISA. TDB and TDM, which are
Mincle ligands,[5] were used as a positive control in the assay.
Recently, Lang and co-workers[22] demonstrated IL-6 production
by stimulation of isolated APCs with TDM and TDB. The
experiments reported here use PBMCs and thus the IL-6 levels
are lower than reported in the literature for these Mincle
ligands. In addition, the solvent used for plate coating greatly
influences the responsiveness of PBMCs to these compounds
(data not shown). Here, we used ethanol as the solvent whereas
other reports in the literature have used isopropyl alcohol or
other solvents. Ethanol was selected to maximize the in vitro
activity of our synthetic compounds and may not be optimal
Figure 2. Cytokine production of UM1024, TDB, TDM, UM1088, 5a–k. Cytokine production from primary human mononuclear cells in response to stimulation
with synthetic trehalose diamide compounds. A) and B) The indicated compound was dissolved in ethanol, serially diluted, and then dried to the bottom of a
°
tissue culture plate. Fresh PBMCs were purified and applied to the compound-coated plates, and the plates were incubated at 37 C; the supernatant was
harvested 24 h later. C) and D) Selected IL-6-positive compounds were subsequently analyzed for TNF, IL-1β by ELISA. Graphs are mean values from 3 donors
�SEM.
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hydrocarbon chain, methyl (5c), ethyl (5a), and pentyl (5f),
gave little to no response. In contrast, 3,4,5-triether compound
(5g) resulted in modest production of IL-6. From our previous
studies in synthesizing the aryl trehalose compounds, ana-
logues with methyl or more importantly a trifluoromethyl group
at the 2-position of the aromatic ring resulted in strong IL-6
production from human PBMCs[21] but with amide linkage, the
compound containing trifluoromethyl group at 2- position (5h
and 5j) had little to no IL-6 production from human PBMCs.
Additionally, it was found that moving the tert-butyl group to
the 5-position (5b) resulted in a loss of activity.
In addition to IL-6, we investigated the production of the
inflammatory cytokines TNF and IL-1β using the IL-6 active
compounds (5g, 5i and 5k) to determine if indeed we are
potentially driving a Th1 or Th17 biased adaptive immune
response. As illustrated, these compounds produce appreciable
quantities of TNF and IL-1β (Figure 2c and d). 5i demonstrated
similar cytokine levels and potency to that of reported Th17-
inducing vaccine adjuvant candidate α-TDE compound B16.[8a]
Further investigations into immunomodulatory properties, ad-
juvant activity and mode of action of 5i are currently underway.
Sulfonamides have been a central functional group in
molecules with a wide spectrum of biological activities.[24] With
the observation that incorporation of the amide linker poten-
tially improved activity versus an ester linkage we decided to
introduce a very polar sulfonamide functional group for the
preparation of a diverse selection of new compounds (Table 2).
The synthesis of sulfonamide analogues was easily investigated
using commercially available sulfonyl chlorides and our
advanced intermediate 6,6’-amine (3). The free base form of
trehalose diamine was generated in situ using solid NaHCO3 and
reacted with aryl sulfonyl chlorides in CH2Cl2 solution in the
presence of Et3N to provided amide intermediates in good to
excellent yields.[25] Standard global benzoyl deprotection with
NaOMe/MeOH gave the target compounds (7a–i) as solids in
good yields (Table 2). The compounds were fully characterized
1
by H,13C NMR, and high-resolution mass spectrometry. From
previous work and the trehalose amide derivatives presented
here, we hypothesized that Mincle activity was very sensitive to
aryl substitution patterns and a high degree of hydrophobicity
may not be required to maintain activity, but a high degree of
steric bulk at the 3- and 5-positions markedly improves IL-6
cytokine production. Therefore, the sulfonyl linked analogue of
UM1088 and 5k were prepared. The 3,5 tert-butyl substituted
aryl sulfonyl chlorides are not commercially available but we
were able to synthesize these using the reported method of
Huntress hese sulfonyl chlorides were then used in coupling
reactions under optimized reaction conditions followed by
deprotection to isolate the derivatives. After several attempts,
we were not able to isolate the corresponding sulfonamide
analogue of UM1088 but we were able to synthesize 7h, the
sulfonamide analogue of 5k. These compounds were assayed
for the secretion of inflammatory cytokines TNF, IL-1β, and IL-6
in human PBMC’s.[5] Surprisingly, compounds bearing the
sulfonamide functional group did not promote IL-6 production
from human PBMCs (Figure 3) with the exception of low- level
activity observed for plate coated 7h and 7i. The response was
verified through the observed mild production of TNF and IL-1β
only at the high concentrations.
To confirm the demonstrated cytokine activity was Mincle
specific the compounds were evaluated for their ability to
signal through human Mincle using human embryonic kidney
Table 2. Synthesis of a focused library of 6,6’-diaryl sulfonamide trehalose compounds.
Compound
Aryl
Yield [%]
Coupling
product
Compound
Aryl
Yield [%]
Coupling
product
Deprotection
Deprotection
7a
7b
76
63
77
71
7f
72
72
80
83
7g
7c
71
80
7h
7i
62
60
69
65
7d
7e
68
65
78
81
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Figure 3. Cytokine production of UM1088, TDB, TDM, and 7a–i. Cytokine
production from primary human mononuclear cells in response to
stimulation with synthetic trehalose diamide compounds. A) The indicated
compound was dissolved in ethanol, serially diluted, and then dried to the
bottom of a tissue culture plate. Fresh PBMCs were purified and applied to
Figure 4. Activation of human Mincle in response to TDM, TDB, and
synthesized trehalose 6,6’-diaryl amide trehalose derivatives. A) and B) The
indicated compounds were plate coated, and hMincle HEK NF-kB-SEAP
reporter cells were incubated with the compounds for 24 h followed by
assessment of the supernatants for SEAP levels. C) Results in HEK-null cells
Data are represented as fold change in OD650 over vehicle-treated cells.
Graphs are mean values from three independent experiments � SEM.
°
the compound-coated plates, and the plates were incubated at 37 C; the
supernatant was harvested 24 h later. B) and C) Selected IL-6-positive
compounds were subsequently analyzed for TNF, IL-1β by ELISA. Graphs are
mean values from 3 donors �SEM.
ability of sulfonamide to signal through hMincle was lower than
their amide counterparts. Further investigations into immuno-
modulatory properties and mode of action of these TDAs are
currently underway.
293 (HEK) cells expressing the human Mincle receptor along
with an NF-kB-driven secreted embryonic alkaline phosphatase
(SEAP) reporter (see the Supporting Information for details).
Several of the analogues were observed to induce the
production of SEAP in a dose-dependent manner. HEK null cells
(HEK cells containing the NF-kB reporter without Mincle
receptor) were used as negative controls to confirm receptor
specificity of the compounds (Figure 4). In a similar fashion to
UM1024, UM1088 demonstrated a strong dose-response in the
hMincle HEK reporter cells. Similarly, 5a and 5c showed a
moderate dose-dependent increase in SEAP production from
hMincle HEK cells.
The rest of the compounds with different substitutions on
the aromatic ring were unable to induce measurable SEAP
production from hMincle HEK reporter cells. Surprisingly, several
of the sulfonamide analogues were able to induce the
production of SEAP in a dose-dependent manner. However, the
In summary, the systematic expansion of our recent report
on the synthesis and evaluation of the novel and very potent
Mincle active compound UM1024 has demonstrated the
exchange of an amide for ester linker markedly improved
responses for the most active compounds. However, incorpo-
ration of the sulfonamide in this scaffold appears to be
deleterious to Mincle activity. This study reinforces our previous
findings that a high degree of steric bulk at the 3- and 5-
positions drastically improves Mincle specific cytokine produc-
tion specifically for the ester and amide scaffolds. While it was
surprising that analogues bearing the sulfonamide were
relatively inactive this finding suggests a crucial interaction of
the linker in the receptor and we intend to further the
understanding of these interactions using molecular dynamics
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Acknowledgements
This work was supported by a NIAID Adjuvant Discovery Program
Contract (HHSN272201400050C). The authors acknowledge Uni-
versity of Montana Core Services of the Center for Biomolecular
Structure and Dynamics (CBSD) and the Department of Chemistry
and Biochemistry supported by the National Institutes of General
Medical Science (NIH CoBRE grant P20GM103546) for NMR and
MS instrumentation. The authors acknowledge the University of
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Conflict of Interest
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The authors declare no conflict of interest.
Keywords: adjuvants · Mincle · TH-17 · trehalose tuberculosis ·
vaccines
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Manuscript received: September 30, 2020
Revised manuscript received: January 6, 2021
Accepted manuscript online: January 7, 2021
Version of record online: February 9, 2021
ChemMedChem 2021, 16, 1246–1251
www.chemmedchem.org
1251
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