Design and Synthesis of N1-Modified Imidazoquinoline Agonists for Selective Activation of Toll-like Receptors 7 and 8

Article abstract of DOI:10.1021/acsmedchemlett.7b00256

A series of N1-modified imidazoquinolines were synthesized and screened for Toll-like receptors (TLR) 7 and 8 activities to identify recognition elements that confer high affinity binding and selectivity. These receptors are key targets in the development of immunomodulatory agents that signal the NF-κB mediated transcription of pro-inflammatory chemokines and cytokines. Results are presented showing both TLR7/8 activations are highly correlated to N1-substitution, with TLR8 selectivity achieved through inclusion of an ethyl-, propyl-, or butylamino group at this position. While the structure-activity relationship analysis indicates TLR7 activity is less sensitive to N1-modification, extension of the aminoalkyl chain length to pentyl and p-methylbenzyl elicited high affinity TLR7 binding. Cytokine profiles are also reported that show the pure TLR8 agonist [4-amino-2-butyl-1-(2-aminoethyl)-7-methoxycarbonyl-1H-imidazo[4,5-c]quinoline] induces higher levels of IL-1β, IL-12, and IFNγ when compared with TLR7 selective or mixed TLR7/8 agonists. The results are consistent with previous work suggesting TLR8 agonists are Th1 polarizing and may help promote cell-mediated immunity.

Full text of DOI:10.1021/acsmedchemlett.7b00256

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Letter
Design and Synthesis of N1-Modified Imidazoquinoline
Agonists for Selective Activation of Toll-like Receptor 7 and 8
Peter Larson, Tamara A. Kucaba, Zhengming Xiong, Michael Olin, Thomas S. Griffith, and David M. Ferguson
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00256 • Publication Date (Web): 16 Oct 2017
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ACS Medicinal Chemistry Letters
Design and Synthesis of N1-Modified Imidazoquinoline Agonists for
Selective Activation of Toll-like Receptor 7 and 8
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Peter Larson,1 Tamara A. Kucaba2, Zhengming Xiong,3 Michael Olin,3,4 Thomas S. Griffith2,4,5,6, and
David M. Ferguson1,7*
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1Department of Medicinal Chemistry, 2Department of Urology, 3Department of Pediatrics, 4Masonic Cancer Center, 5Center
for Immunology, 6Microbiology, Immunology, and Cancer Biology Graduate Program, 7Center for Drug Design, University
of Minnesota, Minneapolis, MN 55455
ABSTRACT: A series of N1-modified imidazoquinolines were synthesized and screened for Toll-like receptor (TLR) 7 and 8 activity
to identify recognition elements that confer high affinity binding and selectivity. These receptors are key targets in the development
of immunomodulatory agents that signal the NF-B mediated transcription of pro-inflammatory chemokines and cytokines. Results
are presented showing both TLR7/8 activation is highly correlated to N1-substitution, with TLR8 selectivity achieved through inclu-
sion of an ethyl-, propyl-, or butylamino group at this position. While the SAR analysis indicates TLR7 activity is less sensitive to
N1-modification, extension of the aminoalkyl chain length to pentyl and p-methylbenzyl elicited high affinity TLR7 binding. Cyto-
kine profiles are also reported that show the pure TLR8 agonist [4-amino-2-butyl-1-(2-aminoethyl)-7-methoxycarbonyl-1H-imid-
azo[4,5-c]quinoline] induces higher levels of IL-1, IL-12, and IFN when compared with TLR7 selective or mixed TLR7/8 agonists.
The results are consistent with previous work suggesting TLR8 agonists are Th1 polarizing and may help promote cell-mediated
immunity.
Keywords: Toll-like receptor, TLR7, TLR8, imidazoquinoline,
SAR, cytokine
The connection between Toll-like receptor (TLR) 7 and
TLR8 activation and cytokine stimulation is well documented.
These endosomal transmembrane receptors recognize and bind
pathogen-associated single stranded RNA and signal (through
the adapter protein MyD88) the NF-B-mediated transcription
of proinflammatory cytokines. This process is critical for
mounting a robust immune response to pathogens by promoting
dendritic cell (DC) maturation, T cell differentiation, B cell dif-
ferentiation and class switching, antibody production, and other
antigen specific responses. These receptors were also found to
recognize small molecule nucleoside base analogs. In seminal
work on TLR7/8 function, Hemmi et al. directly linked the cy-
tokine inductive effects of imiquimod (1), an imidazoquinoline,
with TLR7 activation.1 In subsequent work by Jurk et al., an-
other imidazoquinoline, resiquimod (2) was shown to be a
Figure 1. Heterocycles showing selective and mixed TLR7
and TLR8 activity.
mixed agonist, activating both TLR7 and TLR8.2 Imiquimod,
the prototypical TLR7 selective agonist, was commercialized in
1997 (prior to elucidating its mechanism of action) as a single
agent topical ointment for treating basal and squamous cell car-
cinoma and genital warts, demonstrating the potential utility of
small molecule immunostimulatory agents in treating disease.
tokine profiles and target different cell types. TLR8, for exam-
ple, is functional on myeloid dendritic cells and produces Th1-
polarizing effects which may have significant benefits for the
development of cancer vaccines.3 TLR8 activation also corre-
lates with enhanced cytokine production when compared to
TLR7 selective agents and to the induction of higher levels of
TNF and IFN, consistent with the role of TLR8 in Th1 dif-
ferentiation.4,5 The first TLR8 selective agonist was described
Although TLR7 and TLR8 recognize similar molecular pat-
terns and share common epitopes in binding small molecules,
evidence has emerged in recent years suggesting they are func-
tionally different. TLR7 and TLR8 agonists induce different cy-
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by Gorden et al. in 2005 to study TLR7/8 functional differ-
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Scheme 1. Imidazoquinoline synthesis.a
ences.3 The compound, 3M-002 (3), contains a monoalkyl sub-
stitution to the C2-position of the thiozoloquinoline ring. Alt-
hough no details regarding the design of 3M-002 were given, it
is reasonable to conclude the diminished TLR7 activity dis-
played by this compound was due to the absence of the N1-iso-
butyl group (typical of TLR7 active agents). In 2013, Kokatla
et al. reported the design of a monoalkyl furoquinoline (4) with
high selectivity for TLR8 (and no measurable TLR7 activity).6
Using rational design strategies and X-ray crystallographic
data, this tricyclic scaffold was subsequently refined to yield
three additional classes of TLR8 selective agents, including bi-
cyclic aminoquinolines and benzamidazoles, and substituted
aminoimidazoles.7-9
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A common feature shared by many TLR8 selective imidazo-
quinolines is an alkyl substitution to the A-ring. An SAR study
of imidazoquinolines found activation of both TLR7 and TLR8
increases with C2 alkyl chain length and peaks with a butyl at
TLR7 and a pentyl at TLR8.4,5 These results are partially ex-
plained by the X-ray crystal structure of resiquimod bound to
the TLR8 homodimer complex that shows the C2 substituent
projects into a hydrophobic pocket formed by aliphatic residues
in the receptor.6 The X-ray structure also revealed specific con-
tacts between the N3-nitrogen and 4-amino group with aspartate
and threonine residues of the receptor.10 These data support the
general hypothesis that a hydrogen bond acceptor at position 3
is important to maintain high affinity binding at both TLR7 and
TLR8 (as homology modeling and sequence analysis indicates
these contacts may be conserved).11 Our previous work indi-
cated a hydrogen-bonding donor at the N1 position may also
favor TLR8 activation.4 The SAR data showed a correlation
between TLR8 potency and the presence of an N1-alkylhy-
droxyl group. Since the emphasis of that study was on the C2-
position and alkyl chain length, the N1 substitutions were lim-
ited to short hydroxyalkyl groups.
aReagents and conditions: (i) NEt3, THF, reflux to rt, 18 h, 20–79%;
(ii) CH2I2, isopentyl nitrite, CHCl3, 80 °C, 1 h, 8–48%; (iii)
Pd(PPh3)2Cl2, K2CO3, THF-H2O; (iv) 4N HCl in dioxane (excess),
100 °C. trace–40% yield over two steps. For 12c: (v) 12b, DCC,
DMAP, CH3COOH, CH2Cl2, rt, 1 h, 32%. For 12d: (vi) 12b, DCC,
DMAP, CH3(CH2)6COOH, CH2Cl2, rt, 1h, 52%.
the reaction produced lower yields (<20%) in the presence of a
proton donating group at the N1 position. The methylesters 9u-
w were strategically saponified using potassium hydroxide in
THF-H2O (rt) to liberate the carboxylic acids prior to installa-
tion of the arylester. The Iodo-imidazoles 9a-w were subse-
quently cross coupled with 2-amino-4-methoxycarbon-
ylphenylboronic acid (10) using the Suzuki-Miyaura reaction.
Inefficiencies were noted in the cross coupling where solubility
or steric effects played a significant role. The synthesis of 11k
proved especially problematic with the standard tri-
phenylphosphine ligand. Yields suggested there was less than
one successful turnover of the catalytic cycle even while all the
iodo-imidazole 9k was depleted from the reaction mixture.
Changing the ligand to 2-dicyclohexylphosphino-2′,6′- di-
methoxybiphenyl (SPhos) and the base to cesium carbonate
provided substantially better yields. Impure intermediates 11a-
w were taken directly on to cyclization using anhydrous HCl in
dioxane affording imidazoquinolines 12a-w in trace-40%
yields. The cyclization was also hindered by competing inter-
molecular cross coupling reactions in the presence of N1 pri-
mary amines.
In this study, we expand our original SAR analysis to include
a more diverse set of N1-substitutions (including N1 thiols,
amines, alcohols, esters, and carboxylic acids) to further ex-
plore the structural epitopes that confer TLR7/8 activation and
selectivity. The series design starts with the N1 hydroxyalkyl
analogs identified in our previous work bearing both C2-butyl
and C7-methylester functionalities. The synthetic strategy is
shown in scheme 1 and begins with a multicomponent pyrolysis
reaction to obtain the appropriately N1-substituted imidazoles
(8a-w).12,13 This reaction produced reasonably good yields
(~50-80%) for most N1 substituents, but proved much less effi-
cient when the primary amine was poorly soluble in tetrahydro-
furan (especially 7k-o, u, v). In most cases, yields approaching
30% were afforded by changes to the solvent conditions or the
use of HCl salts. In some cases, the addition of excess TEA
proved beneficial in further improving yields. However, a de-
tailed analysis of all problematic substrates was not performed
so it is unclear if this effect was from proton scavenging or
simply due to increased reaction temperatures. The boc-protect-
ing group of the amines (7p-t) was ultimately removed in situ
during the final acid catalyzed cyclization step. Conversion of
the 5-amino group of the imidazole (8a-w) to the desired iodo
product was accomplished using a modified Sandmeyer reac-
tion.14 As expected from our earlier work using this pathway,
The compounds were evaluated for TLR7/8 activity using
HEK cells expressing TLR7 or TLR8 and the NF-B/SEAP (se-
creted embryonic alkaline phosphatase) gene reporter system.
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This assay measures agonist potency in triggering the down-
stream expression of SEAP mediated by NF-B, which is sub-
Table 1. TLR7 and TLR8 agonist activities.a___________
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sequently quantified using colorimetry. It is important to point
out this assay is not a direct measure of receptor activation or
ligand binding affinity. In addition, some compounds are cyto-
toxic, yielding low optical densities and incomplete dose re-
sponse curves. The results, given in Table 1, show several sub-
stitution-specific trends in TLR7 and TLR8 activity. Prior work
by our lab has shown the presence of an alkyl hydroxyl group
at the N1 position enhanced TLR8 activation. The importance
of this proton donating group at this position is further validated
here by esterification of the 2-hydroxypropyl group of 12b,
yielding TLR8 inactive analogs 12c and 12d. It is noteworthy
to add that the less bulky acetate derivative 12c retained TLR7
activity while the bulkier octanoate derivative 12d was com-
pletely inactive. The results presented here indicate the alkyl
chain length and position of hydroxyl group may be critical as
well given the failure of the hydroxypropyl and hydroxybutyl
derivatives to trigger TLR8.
TLR-7 (mM)
11.4 ± 4.5
1.34 ± 0.26
TLR-8 (mM)
N/A
Compound
R
1
2
6.13 ± 0.26
9
12ab
12bb
1.69 ± 0.11
2.22 ± 0.08
20.86 ± 2.28
9.88 ± 2.13
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12c
12d
3.06 ± 0.34
N/A
N/A
N/A
12eb
12f
14.37 ± 0.63
1.72 ± 0.23
3.21 ± 0.38
2.89 ± 0.28
3.84 ± 0.38
6.67 ± 1.71
3.33 ± 0.46
3.64 ± 0.74
4.53 ± 1.06
1.74 ± 0.88
2.89 ± 0.61
N/A
37.00 ± 2.51
N/Ac
N/A
In contrast to the hydroxyl derivatives, the aminoalkyls (12p-
t) show a positive correlation with increasing alkyl chain length,
with the maximum occurring for the aminopentyl substitution.
This SAR may be in part explained by an X-ray crystallographic
structure reported by Beesu et al. that shows the terminal amino
group of an N1-p-aminomethylbenzyl-substituted imdizoquin-
oline (similar to 12t) extends into a pocket within the TLR8 ec-
todomain and formed favorable hydrogen bonding interactions
with the mainchain carbonyl group of glycine 351.7 It is reason-
able to conclude that the correlation in chain length and TLR8
activity reported here for the aminoalkyl-substituted analogs
may also be due to this interaction. Although X-ray crystallo-
graphic data has indicated another TLR8 residue, Asp545, may
also play a role in stabilizing binding through salt link formation
with terminal amino groups of alkyl substituted ligands,7 this
interaction may not be a contributing factor to the trend dis-
played here. The Asp545 residue is replaced by Leu in the
TLR7 sequence. The potent activity of 12s and 12t in activating
TLR7 suggests an alternate site exists for recognition of the
amino group. The finding that the carboxylates (12u-12w) are
devoid of appreciable TLR7 activity further supports the argu-
ment that an alternative cationic binding site may exist within
the pocket of TLR7 (and perhaps TLR8) for recognition of the
amino group.
12g
12h
12i
N/Ac
N/A
12j
N/A
12k
12l
N/A
N/A
12m
12n
12o
12p
12q
12r
12s
N/A
N/A
N/A
49.8 ± 11.0
26.7 ± 13.9
3.85 ± 0.85
2.21 ± 0.31
N/A
N/A
0.60 ± .13
The remaining compounds in the series show similar activity
in activating TLR7 with little correlation to N1 substitution,
supporting the general conclusion that the structural require-
ments to activate TLR7 are less stringent than TLR8. The ac-
tivity of the bulky methyl esters (12n and 12o) and sulfides (12i
and 12j) suggests the binding site in TLR7 is more tolerant to
aliphatic substitutions. However, the compounds showing the
highest affinity to both TLR7 and TLR8 contain a proton do-
nating group at the N1 position, suggesting this is a primary an-
chor point in the design of next generation analogs with im-
proved potency and selectivity. Given the location of these re-
ceptors within the endosome, it may also be important to in-
clude cell permeability as a screening tool. An analysis of cal-
culated logP values (reported in the SI) shows that the majority
of compounds fall in the range of from 2 to 4. The outliers are
12t
0.18 ± 0.01
190 ± 44
5.34 ± 0.83
N/A
12u
12v
N/A
N/A
12w
N/A
N/A
aCalculated average EC50 based on a minimum of two inde-
pendent measurements performed in triplicate using the
HEK-NF-kB-SEAP-hTLR7 or –hTLR8 reporter cell lines.
c
N/A = no activity at 180mM. bRef. 4. Low optical density.
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the highly lipophilic octanoate derivative 12d (logP = 5.41) and
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the short, polar aminoethyl substituted analog 12p (logP =
2
1.54).
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We next tested the ability of the TLR7 selective compound
12h, the TLR8 selective compound 12p, the TLR7/8 mixed ag-
onist 12t, and the prototypical TLR7 selective agent imiquimod
(1) to stimulate cytokine production from human peripheral
blood mononuclear cells (PBMC). This was accomplished by
measuring the amount of IL-1, IL-12p70, IFN, and TNF by
ELISA after adding titrated doses (50-0.39 mM) of each agonist
to 5 x 105 PBMC. The data (shown in Figure 2) indicate the
TLR8 active compounds produce higher peak cytokine levels
when compared to TLR7 selective compounds. The TLR8 se-
lective agonist 12p was the most potent inducer of IL-1, IL-
12p70, and IFN, while the TLR7/8 mixed agonist 12t stimu-
lated the most TNF production in terms of the peak amount
production for each cytokine. Both compounds reached maxi-
mum responses at approximately 12.5mM. In comparison, the
TLR7 selective agonist 12h showed moderate activity with one
exception, IL-12p70. This compound reached peak activity at
approximately 1 mM and was close in magnitude to 12t in stim-
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ulating this cytokine.
The prototypical TLR7 agonist
imiquimod showed no significant cytokine-inducing activity at
the concentrations tested. The cytokine induction profiles for
each agonist were also quite distinct. The most potent com-
pounds induced sharper peaks. The significance of this result is
not clear but may be due to cell toxicity or downregulation of
TLR signaling at higher ligand concentrations.
The results also suggest TLR7 and TLR8 activity may not be
the only factor involved in triggering cytokine production.
While the data shows TLR8 activity correlates with high cyto-
kine induction, the most potent inducer of IL-1, 12p, is ap-
proximately 6-fold less active than 12t as a TLR8 agonist as
determined by the TLR7/8-HEK reporter system. IL-1 is a key
cytokine in ramping up the adaptive immune response to anti-
gens and is critical to the development of long term immunity.
Production of this cytokine is mediated by the NLRP3 inflam-
masome (a multi-protein complex also activated by pathogen or
danger associated molecular patterns). Although it is not clear
to what extent 12p directly activates the inflammasome, the re-
sults presented here and in previous work reported by our labor-
atory suggest this pathway may be involved in the production
of high IL-1 levels produced by some TLR ligands.5 The ac-
tivity of the TLR7 selective agent 12h in producing IL-12p70
in the low mM range is also noteworthy. IL-12 is Th1 polarizing
and critical to the generation of NK cells, cytotoxic T cells, and
release of IFN and TNF from T cells. The result suggests
some TLR7 selective agents may also be functional in promot-
ing Th1 differentiation and the generation of antigen specific
cellular responses. However, it is important to point out that we
have not evaluated the production of cytokines associated with
Th2 differentiation (e.g. IL-4), so it is not possible to conclude
that this effect is selective.
Figure 2. hPBMC cytokine production measured in triplicate fol-
lowing treatment with selective and mixed TLR7/8 agonists.
In summary, this data presented herein show TLR7 and TLR8
selectivity is sensitive to substitutions to the N1 position of the
imidazoquinoline scaffold. Consistent with prior SAR work by
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our group and others, TLR8 activity was found to correlate with
a hydrogen bonding donor at the N1 position, with the most po-
1
2
tent and selective compounds containing a terminal amino
group. Our results also show TLR7 affinity significantly in-
creases with longer N1 chain lengths containing a terminal
amine. The TLR7 crystallographic structure.15 however, pro-
vided no insight to the structural basis for the high TLR7 po-
tency of 12s and 12t. An analysis of the residues within 6 Å of
the N1 position of resiquimod within the X-ray structure re-
vealed no docking points for a charged amine, suggesting the
agonists may induce changes in the receptor binding site or may
adopt a different orientation. The potent activity of the TLR8
active compounds in producing IL-1, IL-12p70, TNF, and
IFN is also consistent with prior work suggesting TLR8 acti-
vation is Th1 polarizing. These four cytokines are critical for
the production of cell-mediated immunity and Th1 differentia-
tion. The data further suggest pure TLR8 agonists are more po-
tent stimulators of cytokine production than TLR7 or mixed
TLR7/8 agonists. The potent activity of some agonists in pro-
moting the release of specific cytokines raises additional ques-
tions regarding the existence of alternative pathways that may
be activated either directly or indirectly by some ligands. In par-
ticular, the high levels of IL-1 induced by 12p may point to
the recruitment of the NLRP3 inflammasome as a second dan-
ger message sent by the agonist. It is interesting to note that
another study has shown that inert TLR7 ligands are also capa-
ble of enhancing cytokine production when coupled to peptide
antigens.16,17 The mechanism by which these inactive analogs
function, however, is not yet known. More work is therefore
required to understand the role of alternative pathways in the
production of cytokines and to what extent these pathways or
mechanisms can be exploited in the design of cytokine-selective
inductors that avoid the wholesale expression of pro-inflamma-
tory cytokines triggered by TLR-activation. Given that one of
the main limitations to the use TLR-based immunotherapies is
cytokine-associated toxicity, the results presented here are note-
worthy and deserving of additional investigation.
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REFERENCES
(1) 1. Hemmi, H.; Kaisho, T.; Takeuchi, O.; Sato, S.; Sanjo, H.;
Hoshino, K.; Horiuchi, T.; Tomizawa, H.; Takeda, K.; Akira,
S. Small antiviral compounds activate immune cells via the
TLR7MyD88-dependent signaling pathway. Nat. Immunol.
2002, 3, 196–200.
(2) 2. Jurk, M.; Heil, F.; Vollmer, J.; Schetter, C.; Krieg, A. M.
Wagner, H.; Lipford, G.; Bauer, S. Human TLR7 or TLR8 in-
dependently confer responsiveness to the antiviral compound
R-848. Nat. Immunol. 2002, 3, 499.
(3) 3. Gorden, K. B.; Gorski, K. S.; Gibson, S. J.; Kedl, R. M.;
Keiper, W. C.; Qiu, X.; Tomai, M. A.; Alkan, S. S., Vasilakos,
J. P. Synthetic TLR agonists reveal functional differences be-
tween human TLR7 and TLR8. J. Immunol. 2005, 174, 1259–
1268.
(4) 4. Schiaffo, C. E.; Shi, C.; Xiong, Z.; Olin, M.; Ohlfest, J. R.;
Aldrich, C.A.; Ferguson, D. M. Structure–Activity Relation-
ship Analysis of Imidazoquinolines with Toll-like Receptors 7
and 8 Selectivity and Enhanced Cytokine Induction. J. Med.
Chem. 2014, 57, 339-47.
(5) 5. Shi, C. X., Z.; Chittepu, P.; Aldrich, C.C.; Ohlfest, J.R.; and
Ferguson, D.F. Discovery of human toll-like receptor 7/8
ligand analogs with novel cytokine induction. ACS Med.
Chem. Lett. 2012, 3, 501-504.
(6) 6. Kokatla, H. P.; Sil, D.; Malladi, S. S.; Balakrishna, R.; Her-
manson, A. R.; Fox, L. M.; Wang, X.; Dixit, A.; David, S. A.
Exquisite Selectivity for Human Toll-Like Receptor 8 in Sub-
stituted Furo[2,3-c]quinolones. J. Med. Chem. 2013, 56, 6871-
6885.
(7) 7. Beesu, M.; Caruso, G.; Salyer, A. C. D.; Khetani, K. K.; Sil,
D.; Weerasinghe, M.; Tanji, H.; Ohto, U.; Shimizu, T.; David,
S. A. Structure-Based Design of Human TLR8-Specific Ago-
nists with Augmented Potency and Adjuvanticity. J. Med.
Chem. 2015, 58, 7833-7849
(8) 8. Beesu,M.; Malladi,S. S.; Fox, L. M.; Jones, C. D.; Dixit, A.;
David, S. A. Human Toll-Like Receptor 8-Selective Agonistic
Activities in 1-Alkyl-1H-benzimidazol-2-amines J. Med.
Chem. 2014, 57, 7325-7341.
(9) 9. Beesu, M.; Caruso, G,; Salyer, A. C. D.; Shukla, N. M.;
Khetani, K. K.; Smith, L. J.; Fox, L. M.; Tanji, H.; Ohto, U.;
Shimizu, T.; David, S. A. Identification of a Human Toll-Like
Receptor (TLR) 8-Specific Agonist and a Functional Pan-TLR
Inhibitor in 2-Aminoimidazoles. J. Med. Chem. 2016, 59,
3311-3330.
(10) 10. Tanji, H.; Ohto, U.; Shibata, T.; Miyake, K.; Shimizu, T.
Structural reorganization of the Toll-like receptor 8 dimer in-
duced by agonistic ligands. Science, 2013, 339, 1426-1429.
(11) 11. Gentile , F.; Deriu, M.A.; Licandro, G.; Prunotto, A.;
Danani, A.; Tuszynski, J. A. Structure Based Modeling of
Small Molecules Binding to the TLR7 by Atomistic Level Sim-
ulations. Molecules, 2015, 20, 8316-8340.
(12) Ruske, W.; Ruske, E. Nitromalonsäure-dinitril. Chem. Ber.
1958, 91, 2505–2512.
(13) Peinador, C.; Quintela, J. M.; Moreira, M. J. A short and facile
synthesis of Heteromine A. Tetrahedron, 1997, 53, 8269–
8272.
(14) Minakawa, N.; Kojima, N.; Matsuda, A. Nucleosides and nu-
cleotides. 184. Synthesis and conformational investigation of
anti-fixed 3-deaza-3-halopurine ribonucleosides. J. Org.
Chem. 1999, 64, 7158–7172.
(15) Zhang, Z.; Ohto, U.; Shibata, T.; Krayukhina, E.; Taoka,
M.; Yamauchi, Y.; Tanji, H.; Isobe, T.; Uchiyama,
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ASSOCIATED CONTENT
Supporting Information. Full experimental details and compound
characterization data as well as biological methods and procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 612-626-2601. E-mail: ferguson@umn.edu.
Funding
Support for this study, in part, was provided by the Masonic
Cancer Center, University of Minnesota (to TSG) and the
Randy Shaver Cancer Research & Community Fund (to TSG).
ABBREVIATIONS
TLR, toll-like receptor; NF-κB, nuclear factor kappa-light-chain-
enhancer of activated B cells; IFN, interferon; PBMC, peripheral
blood mononuclear cell; IL, interleukin; TNF, tumor necrosis fac-
tor.
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S.; Miyake, K.; Shimizu, T. Structural Analysis Reveals that
Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Sin-
gle-Stranded RNA. Immunity 2016, 45, 737-748.
(16) Gao, D.; Liu, Y.; Diao, Y.; Gao, N.; Wang, Z.; Jiang, W.; Jin,
G. Synthesis and Evaluation of Conjugates of Novel TLR7 In-
ert Ligands as Self-Adjuvanting Immunopotentiators. ACS
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Med. Chem. Lett., 2015, 6, 249–253.
(17) Gao, D.; Diao, Y.; Li, W.; Gao, N.; Liu, Y.; Wang, Z.; Jiang,
W.; Jin, G. Toll-Like Receptor 7 Inactive Ligands Enhanced
Cytokine Induction by Conjugation to Weak Antigens.
ChemMedChem. 2015, 10, 977-980.
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Design and Synthesis of N1-Modified Imidazoquinoline Agonists for
Selective Activation of Toll-like Receptor 7 and 8
Peter Larson,1 Tamara A. Kucaba2, Zhengming Xiong,3 Michael Olin,3,4 Thomas S. Griffith2,4,5,6, and David
M. Ferguson1,7*
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1Department of Medicinal Chemistry, Department of Urology, Department of Pediatrics, Masonic
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Cancer Center, Center for Immunology, Microbiology, Immunology, and Cancer Biology Graduate
Program, 7Center for Drug Design, University of Minnesota, Minneapolis, MN 55455
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