Discovery of imidazoquinolines with toll-like receptor 7/8 independent cytokine induction

Article abstract of DOI:10.1021/ml300079e

Toll-like receptors (TLRs) are key targets in the design of immunomodulating agents for use as vaccine adjuvants and anticancer treatments. The imidazoquinolines, imiquimod and resiquimod, have been shown to activate TLR-7 and -8, which in turn induce cytokine production as part of the innate immune response. Herein, we report the synthesis and discovery of a C7-methoxycarbonyl derivative of imiquimod that stimulates cytokine production but is devoid of TLR-7/8 activity. Data are presented that shows that this analogue not only induces IL-12p40 and TNF production, similar to that of imiquimod and resiquimod, but greatly enhances the production of IL-1β, a key cytokine involved in the activation of CD4 T cells. It is further demonstrated that TLR-7/8 activation can be recovered by the addition of a C2-alkyl substituent to this newly discovered analogue. The results support the existence of an alternative mechanism of action by which imidazoquinolines can stimulate cytokine production.

Full text of DOI:10.1021/ml300079e

Letter
pubs.acs.org/acsmedchemlett
Discovery of Imidazoquinolines with Toll-Like Receptor 7/8
Independent Cytokine Induction
Ce Shi,†,∇ Zhengming Xiong,‡,∇ Padmaja Chittepu,† Courtney C. Aldrich,† John R. Ohlfest,‡,§,∥,⊥
and David M. Ferguson*,†,#
†Center for Drug Design, University of Minnesota, 516 Delaware Street SE, Minneapolis, Minnesota 55455, United States
§
∥
⊥
‡Department of Pediatrics, Department of Neurosurgery, Cancer Center, and Stem Cell Institute, University of Minnesota
Medical School, Minneapolis, Minnesota 55455, United States
#Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: Toll-like receptors (TLRs) are key targets in the
design of immunomodulating agents for use as vaccine adjuvants and
anticancer treatments. The imidazoquinolines, imiquimod and
resiquimod, have been shown to activate TLR-7 and -8, which in
turn induce cytokine production as part of the innate immune
response. Herein, we report the synthesis and discovery of a C7-
methoxycarbonyl derivative of imiquimod that stimulates cytokine
production but is devoid of TLR-7/8 activity. Data are presented that
shows that this analogue not only induces IL-12p40 and TNF
production, similar to that of imiquimod and resiquimod, but greatly enhances the production of IL-1β, a key cytokine involved
in the activation of CD4 T cells. It is further demonstrated that TLR-7/8 activation can be recovered by the addition of a C2-
alkyl substituent to this newly discovered analogue. The results support the existence of an alternative mechanism of action by
which imidazoquinolines can stimulate cytokine production.
KEYWORDS: imidazoquinolines, toll-like receptor 7/8, cytokine induction
agonists of this pathway. These compounds first appeared in
the patent literature in the 1980s.5,6 Topical imiquimod cream
was FDA approved in 1997 for the treatment of basal cell
carcinoma, and there remains extensive interest in optimizing
this class of compounds for multiple indications.7,8 Although
their ability to induce interferon-α (IFNα) was known at that
time, the mechanism of action was not discovered until 2002
when Hemmi et al. definitively linked the imidazoquinolines to
TLR 7 function.9 They reported that both 1 and 2 failed to
induce cytokine production in TLR 7-deficient mice. The
results were further supported and extended by Gorden et al.
and Jurk et al. who disclosed that resiquimod also activates the
TLR 8 signaling pathway.10,11
oll-like receptors 7 and 8 (TLR 7/8) are key targets for
Tthe design of small molecule immunomodulators for use
as vaccine adjuvants and anticancer/antiviral agents.1−3 These
receptors trigger the nuclear factor κ light-chain enhancer of
activated B cells (NF-κB) mediated production of proflamma-
tory cytokines and chemokines in response to the presence of
viral single-stranded ribonucleic acid (ssRNA) within the
endosome of B cells and dendritic cells.4 The imidazoquino-
lines imiquimod (1) and resiquimod (2) (Figure 1) are potent
Following the discovery that imidazoquinolines activate
TLR-7 and -8, Gerster et al. reported the synthesis and
evaluation of a large series of analogues that included many
compounds from the original patent literature.12 Although the
compounds were evaluated for IFNα production, TLR 7/8
functional data were not reported. The most potent analogues
were found to contain a short alkyl chain at the C-2 position
(with n-butyl showing the highest potency) and short, hydroxyl
alkyl chains at N-1. In terms of aryl substitutions, it was
Received: April 4, 2012
Accepted: May 12, 2012
Published: May 12, 2012
Figure 1. Imidazoquinolines.
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
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a
determined that the amino group at C-4 was required for
activity. Although the chemistry was limited, simple sub-
stitutions to the remaining aryl positions were found to abolish
activity, except in the case of C-7. The C-7 methoxy, hydroxyl,
and methyl derivatives retained modest IFNα production. In
more recent work, Shukla et al. verified the TLR 7 activity of a
series of C-2 and N-1 substituted imdazoquinolines, including
imiquimod.13 Their results indicated that the most potent and
TLR 7 selective compounds contained an n-butyl group at C-2
(as suggested by Gerster et al.) and either a 2-hydroxy-2-
methylpropyl or a benzyl group at N-1. Although it was
reported that all of the C-2 alkyl compounds were TLR 7-
selective, a systematic evaluation of TLR 8 binding was not
presented.
Scheme 1. Synthesis of Imidazoquinoline Analogues
One of the limiting factors in the design and synthesis of
imidazoquinolines for screening is the cumbersome nature of
the synthetic route. Prior reports have used traditional
heterocyclic chemistry that is borrowed from purine base
synthesis.14 The approach applies a modified Traube reaction
to annulate diaminoquinolines by condensation with ortho
esters or acid chlorides (yielding the desired imidazoquinoline
tricycle). While this approach allows substitutions to be
efficiently introduced to the imidazole ring system, modifica-
tions to the quinoline ring are more complicated, requiring
additional synthetic steps to be added to obtain the substituted
precursors. Functional group modifications must also be
compatible with the reaction conditions used in subsequent
steps, further limiting the synthetic pathway. To address this
problem, we have developed an alternative route that exploits
the Suzuki−Miyaura reaction to efficiently synthesize highly
substituted imidazoquinolines in four steps. In this report, we
describe the application of this chemistry to produce a set of C-
7 methoxy carbonyl derivatives (5 and 6) in addition to
unsubstituted analogues (3 and 4). We also present TLR 7/8
and cytokine screening data that indicates that this series
functions through a unique pathway in activating cytokine
production.
The synthesis of 3−6 (Scheme 1) begins with the
multicomponent condensation of the aminomalononitrile (7),
orthoester (8a/b), and β-amino alcohol (9) to afford the 5-
aminoimidazole-4-carbonitriles (10a/b).15,16 The key iodoimi-
dazole intermediates (11a/b) are obtained in good yield using a
modified Sandmeyer reaction employing isoamyl nitrite and
diiodomethane.17 Suzuki coupling of 11a/b with 2-amino-
phenylboronic acid (12a) or 2-amino-4-methoxycarbonylphe-
nylboronic acid (12b) furnishes the atropisomeric intermedi-
ates (13−16). Intramolecular cyclization is achieved by
treatment of the heterobiaryl intermediates with either
anhydrous HCl or NaOMe to afford the desired imidazoquino-
lines (3−6). Acid catalysis is preferred for substrates 15 and 16
due to partial saponification of the methyl esters under the
basic conditions. Although this approach provides an efficient
method to install novel functionality on the benzene ring of the
quinoline, we found that the efficiency of the cross-coupling
step was influenced by the C-2 alkyl substituent. It was noted
that 11b reacted sluggishly with a significant portion under-
going competitive protio-dehalogenation while 11a furnished
the desired cross-coupled products (13 and 14) in 3 h with
high yields. To avoid steric hindrance with the bulky C-2
substituent, the N-1 group in 11b likely is directed toward the
5-iodo substituent, thereby hindering the palladium-catalyzed
cross-coupling and explaining its lower intrinsic reactivity.
a
Reagents and conditions: (i) NEt3, THF, reflux to rt, 15 h, 65−88%.
(ii) CH2I2, isoamylnitrite, CHCl3, 80 °C, 30 min, 65−75%. (iii)
Pd(OAc)2, PPh3, Na2CO3, DME−H2O, 100 °C, 3−15 h, 34−95%.
(iv) 4 N HCl in dioxane (excess), 80 °C, 85−93%.
Compounds 1−6 were initially screened using TLR 7 and
TLR 8 reporter cell lines for their ability to induce the NF-κB
pathway. Consistent with prior studies, imiquimod (1) was
determined to be a selective TLR 7 agonist, whereas
resiquimod (2) triggered both TLR7 and TLR 8 (as reported
in Table 1). Resiquimod also showed an increased potency at
TLR 7 as compared to that of imiquimod. This increased
potency may be linked to the C-2 substituent as suggested by
the activity of compound 3. Deletion of the C-2 ethoxymethyl
moiety of resiquimod resulted in a 67-fold drop in TLR 7
agonist activity and abolished TLR 8 activation entirely. Further
evidence to support the vital role C-2 substitution plays in both
potency and selectivity can be found in the data reported for 4.
Both TLR 7 and 8 function was restored and enhanced by
addition of a C-2 butyl group to 3. The C-2 butyl substitution
in 4 increased TLR 7 and 8 potency by approximately 10- and
60-fold, respectively, when compared to resiquimod.
The most intriguing and novel findings were revealed when a
methyl ester was added to the quinoline ring at C-7, generating
compound 5. This compound was entirely devoid of TLR 7
and TLR 8 agonist activity as measured by the reporter cells.
This result was completely unexpected. On the basis of
previous reports that indicated that C-7-substituted imidazo-
quinolines stimulated the release of IFNα, the compounds were
further screened for cytokine production. The results given in
Table 1 show that despite displaying no appreciable activity in
triggering TLR 7 or 8, compound 5 induced the release of
interleukin 1β (IL-1β) from mouse bone marrow-derived
dendritic cells (BMDC) at levels 15 times greater than
compounds 1−4. In addition to IL-1β, compound 5 induced
2−3 times more tumor necrosis factor (TNF) α and IL-12p40
than imiquimod, demonstrating a broad induction of
inflammatory cytokines (commonly linked with TLR activa-
tion). Once again, TLR 7 and 8 binding was restored by the
addition of a C-2 butyl group to 5. Although 6 was not the
most potent TLR 7 and 8 agonist of the group, the ability to
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Table 1. TLR 7/8 Agonist Activities of Analogurs 1−6 and Their Corresponding Cytokine Production Data
cytokine (pg/mL)
IL-1β IL-12p40
EC50 (μM)
compd
TNFα
IL-10
33
72 16
60
TLR-7
TLR-8
b
1
593 62
2134 266
2041 31
1743 165
1451 137
1353 552
91.3 34.5
35
38
1
4
889 100
1491 31
839 35
2
12.1 4.4
1.4 0.3
95 20
0.1 0.02
n/a
n/a
2
6.4 2.6
n/a
3
61 18
73 8.2
8
4
864 187
1773 62
1286 276
260.6 11.9
181 24
94 14
110 45
0.100 0.003
n/a
5
1144 116
893 306
26.2 3.7
6
1.2 0.3
41.3 3.6
control
16.2 4.7
a
b
Multiplexed cytokine production was measured in triplicate using analogues at 30 μM. No activation (>270 μM).
significantly enhance IL-1β production was retained, suggesting
the C-7 methyl ester derivatives trigger an alternative pathway
to cytokine production.
ABBREVIATIONS
■
TLR, toll-like receptor; NF-κB, nuclear factor κ light-chain
enhancer of activated B cells; ssRNA, single-stranded
ribonucleic acid; IFN, interferon; BMDC, bone marrow-derived
dendritic cell; CD4, cluster of differentiation 4; IL, interleukin;
TNF, tumor necrosis factor
To our knowledge, this is the first demonstration of an
imidazoquinoline that triggers a profound induction of
inflammatory cytokines in the absence of TLR 7 or 8 agonist
activity. Although the mode of action of 5 is currently not
known, some clues to the pathways involved may be found in
high levels of IL-1β reported for 5 and 6. The unique and
important role of IL-1β has only recently been appreciated as
“signal three” for activation of cluster of differentiation 4 (CD4)
T cells. Specifically, IL-1 receptor-deficient CD4 T cells have
inferior proliferative and functional capacities, demonstrating a
direct effect of IL-1β on the T cell. Moreover, recombinant IL-
1β can profoundly improve memory T cell formation and
antibody responses against protein antigens in mice.18 It is
important to note that IL-1β release is dependent on activation
of the inflammasome, an intracellular pathway that senses
microbial infection including ssRNA.19 Thus, it is reasonable to
conclude that the alternative pathway accessed by the C-7
methyl ester derivatives may involve inflammasome activation.
Considering that IL-1β has a unique capacity to increase
antibody responses and modulate CD4 T cell differentiation,
this discovery could be the first step in developing novel
imidazoquinoline analogues that have superior activity as
vaccine adjuvants or for single agent therapy. This hypothesis
awaits validation in mouse models and human patients.
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ASSOCIATED CONTENT
* Supporting Information
■
S
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.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 612-626-2601. E-mail: ferguson@umn.edu.
■
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Author Contributions
∇These authors contributed equally to this work.
Funding
This work was supported in part by NIH Grants R01
CA154345, R01 CA160782, the American Cancer Society
grant RSG-09-189-01-LIB, and generous support from the
Minnesota Medical Foundation, the Hedberg Family Founda-
tion, and the Children's Cancer Research Fund to J.R.O.
D.M.F. and J.R.O. also thank the Minnesota Futures Grant
Program for financial support of this work.
Notes
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Purines. II. The synthesis of certain purines and the cyclization of
The authors declare no competing financial interest.
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