Structure-activity relationship analysis of imidazoquinolines with toll-like receptors 7 and 8 selectivity and enhanced cytokine induction

Article abstract of DOI:10.1021/jm4004957

Toll-like receptors 7 and 8 (TLRs) have emerged as key targets in the design of small molecule adjuvants and stimulants for use in immunotherapies. This study examines the structure-activity relationship of a series of C2- and N1-substituted C7-methoxycarbonylimidazoquinolines to gain insight to the structural basis to TLR-7 and -8 selective activity. The analysis is further applied to evaluate the induction of multiple cytokines, including IL-10, IL-12, IL-1β, TNF-α, IFN-α, and IFN-γ, using murine BMDCs and human PBMCs. The results show TLR-7/8 activity is correlated to the C2-alkyl chain length, with peak activity occurring for the butyl (TLR-7) and pentyl (TLR-8) derivatives. A similar SAR is identified in the production of IL-1β, IL-12, and IFN-γ, which are shown to depend on both the C2-alkyl chain length and substitution to the N1-position. The compounds were also potent stimulators of IFN-α and IL-10 production but with less pronounced structure-based correlations.

Full text of DOI:10.1021/jm4004957

Article
pubs.acs.org/jmc
Structure−Activity Relationship Analysis of Imidazoquinolines with
Toll-like Receptors 7 and 8 Selectivity and Enhanced Cytokine
Induction
Charles E. Schiaffo,† Ce Shi,† Zhengming Xiong,‡ Michael Olin,‡ John R. Ohlfest,§,⊥
Courtney C. Aldrich,∥ and David M. Ferguson*,†,∥
†Center for Drug Design, University of Minnesota, 516 Delaware Street SE, Minneapolis, Minnesota 55455, United States
‡Department of Hematology and Oncology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, United States
§Department of Pediatrics and Cancer Center, University of Minnesota Medical School, Minneapolis, Minnesota 55455, United
States
∥Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
ABSTRACT: Toll-like receptors 7 and 8 (TLRs) have emerged as key targets in the design of small molecule adjuvants and
stimulants for use in immunotherapies. This study examines the structure−activity relationship of a series of C2- and N1-
substituted C7-methoxycarbonylimidazoquinolines to gain insight to the structural basis to TLR-7 and -8 selective activity. The
analysis is further applied to evaluate the induction of multiple cytokines, including IL-10, IL-12, IL-1β, TNF-α, IFN-α, and IFN-
γ, using murine BMDCs and human PBMCs. The results show TLR-7/8 activity is correlated to the C2-alkyl chain length, with
peak activity occurring for the butyl (TLR-7) and pentyl (TLR-8) derivatives. A similar SAR is identified in the production of IL-
1β, IL-12, and IFN-γ, which are shown to depend on both the C2-alkyl chain length and substitution to the N1-position. The
compounds were also potent stimulators of IFN-α and IL-10 production but with less pronounced structure-based correlations.
Since that time, significant progress has been made in
understanding the molecular basis of the function of these
nucleoside analogues. In 2005, Gerster et al. reported the IFN-
α inductive effects of a large collection of imidazoquinolines
(Figure 1), including imiquimod.7 A structure−activity relation-
ship (SAR) analysis was presented that indicated that increased
IFN-α production could be obtained by addition of short alkyl
substituents to the C2 position of the imidazole ring. The
second generation congener resiquimod (2) contains this
design element and is an extremely potent, dual TLR-7/8
agonist. More detailed studies of receptor activation have
shown that substitution to the C2 position may also have an
impact on receptor selectivity. Shukla et al. reported an SAR
study that evaluated the TLR-7 activity of a variety of C2-
substituted imidazoquinolines, including the highly TLR-7
selective agonist gardiquimod (3).8 A correlation between the
C2-alkyl chain length and activity was reported that identified
the butyl substitution to be optimal for activity. Heteroatom
substitutions to the C2-alkyl chain, such as that found in
INTRODUCTION
■
Toll-like receptors (TLRs) have emerged as key targets in the
design and development of immunomodulating agents for use
as vaccine adjuvants.1,2 These receptors recognize pathogen
associated molecular patterns and signal the release of
proinflammatory cytokines and chemokines. Although at least
10 human TLRs are known to exist, TLR-7 and -8 have become
popular targets for drug discovery, since they recognize small
synthetic molecules.3 Both receptors are located on the
endosomal walls of monocytes, macrophages, and dendritic
cells; TLR-7 and TLR-8 are also expressed on the endosomal
walls of B cells and mast cells, respectively. In all cell types, both
receptors respond to the presence of viral single stranded RNA
(ssRNA) within the cytoplasm.4 Their activation induces the
NF-κB mediated transcription of cytokines and chemokines
through a myeloid differentiation protein 88 (MyD88)
dependent pathway.5,6 The first FDA approved drug that
took advantage of this signaling pathway was topical imiquimod
(1), an imidazoquinoline analogue with potent activity in
stimulating interferon production. Imiquimod first appeared in
the patent literature in 1985 and was approved for the
treatment of basal cell carcinoma in 1997.
Received: July 19, 2013
© XXXX American Chemical Society
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More recently, Shi et al reported an alternative strategy to
obtain highly substituted imidazoquinolines in approximately
four steps.9 A retrosynthetic analysis of the methods is
compared in Figure 2. The approach exploits a Suzuki coupling
reaction to construct a substituted biaryl precursor that is
readily cyclized to the final product under mild conditions. Shi
et al. applied this scheme to functionalize the aryl ring to
include a C7 methyl ester. The lead compound that emerged, 4,
was found to stimulate enhanced levels of cytokine production
using bone marrow derived dendritic cells (BMDCs) as the
model system.9 Moreover, data were presented that directly
linked the C2-alkyl chain substitution to increased TLR-7 and
-8 function for this C7-acylated imidazoqinoline congener. In
this report, we evaluate the TLR-7 and -8 activity and cytokine
induction of a series of C2- and N1-substituted 7-methoxy-
carbonylimidazoquinolines. An SAR analysis is presented to
gain insight to the structural features that confer TLR-7 and -8
binding and selectivity as well as the requirements for
stimulating cytokine production.
Figure 1. Imidazoquinolines.
gardiquimod, did not improve the activity, which is somewhat
surprising given the potency of resiquimod at TLR-8.
Resiquimod and gardiquimod differ by a single heteroatom
substitution in the C2 alkyl chain. Although not elaborated
upon, the data also show that potency may depend on the
substituent on the N1 position with the most potent compound
containing an N1-2-methyl-2-hydroxypropyl group identical to
that of resiquimod.
A limiting factor to the design of SAR studies involving
imidazoquinolines is the synthesis. The traditional route
employs a modified Traube reaction to cyclize the imidazo
ring system from a substituted o-diaminoquinoline. The
approach is outlined in Figure 2 and requires the C4-amino
RESULTS AND DISCUSSION
Chemistry. The synthetic route to obtaining highly
substituted imidazoquinolines is shown in Scheme 1. The 4-
■
a
Scheme 1
Figure 2. Retrosynthetic analysis showing the key differences in
construction of the imidazoquinoline structure. The traditional route
(A) is limited by the use of quinoline building blocks that must be
functionalized early in the reaction sequence. This not only adds
additional steps to the synthesis but limits the complexity of the
substituents to those that can tolerate fairly harsh reaction conditions.
The route reported by Shi et al. (B) applies a biaryl cross-coupling
reaction to construct highly substituted biaryl precursors that are
cyclized under mild conditions, allowing complex functionalities to be
built into the imidazoquinoline structure.
group to be installed as the last step in the sequence by either
rearrangement of an N-oxide or ammonolysis.7 While this
scheme is amenable to modifications to the N1 and C2
positions, the reaction conditions limit substitutions to the C6−
C9 aryl positions to end stage bromination or nitration. The
quinoline precursor can also be modified, but once again, this
approach is limited to those substitutents that can tolerate the
reaction sequence. Gerster et al. examined the IFN inductive
activity of a collection of C6−C9 analogues that were mainly
composed of halo, nitro, hydroxy, methoxy, and methyl
substitutions. Only the C6 hydroxy and C7 methyl, methoxy,
and hydroxyl modifications retained activity. The SAR analysis
presented, however, did not examine the effect of combining
N1 or C2 substitutions with C6−C9 aryl modified analogues.
a
Reagents and conditions: (i) NEt3, THF, reflux to rt, 15 h, 40− 88%;
(ii) CH2I2, isopentyl nitrite, CHCl3, 80 °C, 30 min, 30−90%; (iii)
Pd(PPh3)3Cl2, K2CO3, THF−H2O; (iv) 4 N HCl in dioxane (excess),
100 °C, 20−70% yield over two steps.
cyano-5-iodoimidazole precursors (8a−m) are synthesized
through the multicomponent condensation of aminomalononi-
trile (5), orthoester (6), and β-amino alcohol (7).10,11 In
general, the reaction proceeded in good yield and without
complications. The exception to this was the synthesis of 8g.
Prior work has shown the benzyl orthoester to be remarkably
stable to pyrolysis.12 This problem was overcome by the
B
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ac
,
Table 1. TLR-7 and -8 Activities and Cytokine Induction of Substituted Imidazoquinolines
a
b
c
Multiplexed cytokine production was measured in triplicate using analogues at 30 μM. HEK SEAP reporter assay. n/a indicates no activation.
1
addition of another equivalent of the benzyl orthoester and
extending the initial reaction time to 6 h. The conversion of
8a−m to 9a−m is accomplished using a modified Sandmeyer
reaction employing isoamyl nitrite and diiodomethane. Lower
yields (∼30%) were noted when R2 contained a secondary
alcohol which most likely acted as a proton source.13 The
highest yields (∼90%) were obtained when R1 was
unsubstituted. Suzuki−Miyaura coupling of 9a−m with 2-
amino-4-methoxycarbonylphenylboronic acid (10) provided
intermediates 11a−m, which are directly carried onto the
next step without purification. It is interesting to note that these
heterobiaryl intermediates are axially chiral because of the
hindered rotation about the ortho substituted biaryl bond. The
diasterotopic protons of the methylene unit of 11a are readily
identified in the H NMR as a doublet of doublets with a
geminal coupling constant of 14−15 Hz.9 Cyclization of 11a−
m employing anhydrous HCl in dioxane affords imidazoquino-
lines 12−24 in 20−70% yield over two steps as white to yellow
amorphous solids. The variable yields can be directly attributed
to the efficiency of the biaryl coupling step. Although the
variability in the Suzuki yields was not studied in any systematic
matter, the results suggest that the efficiency may be related to
solubility. The coupling was performed in a mixed solvent
aqueous environment. More hydrophilic iodide 9a proceeded
in higher yield, 67%, while hydrophobic 9l resulted in the
lowest yield, 23%, of all the compounds. The cyclization was
determined to occur in near quantitative yield.
C
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Biological Activity. The analogues (12−25) were screened
for TLR-7 and -8 activity in vitro using HEK-293 cells that were
transfected with either the human TLR-7 or -8 gene along with
the NF-κB inducible SEAP (secreted embryonic alkaline
phosphatase) reporter gene. This assay measures NF-κB
mediated SEAP production spectrophotometrically following
TLR-7 or -8 specific activation. An SAR analysis of the data
given in Table 1 shows some general trends in TLR selective
activity. TLR-7 is clearly more tolerant to substitution than
TLR-8, and a general trend in increased activity is apparent
when the C2 alkyl chain is lengthened from methyl to butyl. A
slight decrease is noted in activity when the alkyl chain length
reaches pentyl and beyond (to benzyl), suggesting that the
butyl is optimal for TLR-7 activity. This general trend is
followed for TLR-8 as well, albeit TLR-8 binding is less tolerant
of substitution, with only the butyl and pentyl retaining activity.
The failure of the C2-unsubstituted 7-ester analogue 12 to
activate TLR-7 as well as TLR-8 is quite interesting and has
been discussed in a previous publication.9 From a comparison
of the structure of 12 with that of imiquimod, the compounds
differ by the addition of the C7-ester and the tertiary alcohol at
N1. The C7 unsubstituted congener of 12, however, selectively
activates TLR-7, similar to that of imiquimod.9 While the data
are limited, the results indicate the C7 ester substitution
decreases binding affinity at TLR-7 and -8 while the addition of
C2-alkyl group restores activity.
A limited series of N1-modified analogues containing the C2-
butyl group and C7-ester functionality are also reported in
Table 1. The results indicate that the 2-hydroxypropyl
stereoisomers (19−21) show the greatest potencies as mixed
TLR-7/8 agonists, with EC50 values similar to that of
resiquimod (which contains an N1-2-methy-2-hydroxypropyl
group). A comparison of the racemate (19) and the S (20) and
R (21) enantiomers reveals no significant differences in TLR-7
and -8 activity, suggesting that receptor binding is not
enantioselective. The des-methyl primary alcohol congener
(22), however, shows a significant reduction in TLR-7 function.
Although the function can be restored by the addition of a
methyl or benzyl ether, the loss of the hydroxyl group at the N1
position has a significant impact on TLR-8 activity. The data
suggest that high affinity binding to TLR-8 may depend on the
presence of a hydrogen bonding donor on the alkyl chain of
N1. TLR-7 activity, on the other hand, correlates well with
increased substitution to the N1-hydroxyalkyl side chain,
including the addition hydrophobic groups (e.g., the benzyl
ether).
including TNF-α, IL-1β, IL-12, and IL-10. IL-1β plays an
important role in activating the adaptive immune system and in
CD4+ T cell health and has been implicated in T cell
memory.18 In general, the results show that IL-1β production
increases as a function of the C2-alkyl chain length and reaches
a maximum for the pentyl derivative. The potency of the C2-
butyl derivatives, however, is shown to improve when the N1 is
modified to a 2-hydroxypropyl or methoxyethyl side chain.
Similar results are observed for IL-12 production. This cytokine
is produced by dendritic cells and leads to the differentiation of
immature CD4+ T cells, the activation and memory formation
of CD8+ T cells, and the activation of NK cells.19 Once again,
the C7-ester analogues are the most potent with peak induction
occurring for the S-configured enantiomer of the C2-2-
hydroxypropyl analogue (20). A further evaluation of the
related stereoisomers (19−21) shows only a slight preference
for the S-isomer, indicating that the induction is not
enantioselective (similar to the TLR-7/8 results). The activity
is reduced, however, when the N1 position is substituted with
the bulky benzoyloxyethyl moiety. IL-12 production also shows
a correlation with C2-alkyl chain length, which reaches a
maximum for the butyl substitution in this case.
The final two cytokines, TNF-α and IL-10, show much
weaker correlations with the N1 and C2 substitutions. IL-10 is
an anti-inflammatory cytokine that stimulates the humoral
immune response including B cell maturation and antibody
production.20 High production of this cytokine, however,
down-regulates the cytotoxic T-cell response to APCs, which
can be detrimental to the efficacy of some types of
immunotherapies (such as cancer vaccines). TNF-α, on the
other hand, is a proinflammatory cytokine and potent
immunostimulant.21 While TNF-α is considered beneficial to
many immunotherapies, high systemic levels can produce
severe toxicity. The results indicate that IL-10 and TNF-α
induction both fail to display the structure-based trends noted
above for IL-1, IL-12, and TLR-7/8 activity. In fact, the most
active IL-10 and TNF-α stimulants are resiquimod and the N1-
benzyloxyethyl derivative (23), respectively, which are both
weak stimulants of IL-1β and IL-12.
The compounds were also evaluated using human peripheral
blood mononuclear cells (PBMCs). This assay captures the
stimulation of cytokine production from multiple cell types,
including dendritic cells, T cells, B cells, monocytes, and natural
killer (NK) cells. The population of these cell types, however,
varies greatly among donors, producing significant variance in
the assay. Figure 3 shows the TNF-α, IFN-α, and IFN-γ
induction derived from three human blood donors. The more
active compounds identified using BMDCs are potent
stimulants of PBMCs as well (e.g., the C2-pentyl (17) and
the secondary alcohols 19−21). The IFN-α levels show the
least variance across the series, which is not surprising, since
this cytokine is secreted by multiple cells types. IFN-α primarily
functions in antiviral responses and activates a cohort of genes
in producing an immune response. IFN-γ, on the other hand, is
immune cell specific and is primarily secreted by NK and T
cells. It functions by up-regulating antigen specific responses in
the immune system (such as antigen presentation) and by
activating macrophages. The IFN-γ data are more consistent
with the structure-based trends established for IL-1β and IL-12
using BMDCs and once again identify the C2-butyl, C2-pentyl,
and N1-2-hydroxypropyl derivatives as potent stimulants. The
TNF-α values show similar structure-based trends with the
exception of resiquimod, which is significantly more potent in
The imidazoquinolines (12−25) were further screened for
cytokine production in BMDCs using a flow cytometry reporter
assay. Dendritic cells are antigen presenting cells (APCs) that
play a crucial role in activating the immune response through
the presentation of antigen to T cells (both CD4+ and CD8+),
expression of ligands for co-stimulatory receptors on T cells,
and release of stimulatory cytokines. It is important to point
out, however, that the function of TLR-8 in triggering an NF-
κB mediated response in the murine system is not fully
understood. While the original reports by Hemmi et al. and
Jurk et al. strongly suggested that the TLR-8 is not functional in
recognizing small molecule agonists, more recent work has
suggested that the receptor plays a role in regulating TLR-7
activation.14−17 Nevertheless, murine BMDCs provide a direct
measure of an immune cell specific response to TLR agonists
and provide a well established model for assaying cytokine
induction. Table 1 reports the production of four key cytokines
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this position, suggesting that TLR-7 may be the “default”
receptor for imidazoquinoline recognition. The idea that certain
receptors within families contain additional selectivity elements
is not new and is known to be important to G-protein-coupled
receptor function and recognition (e.g., the μ, δ, and κ opioid
receptors).22 While this may simply mean that it is more
difficult to selectively activate TLR-8, the finding may have
larger ramifications to the function TLR-7 versus TLR-8 plays
in mediating the innate immune response to viral RNA. The
availability of crystallographic data for the TLRs will have a
great impact on understanding the role of ligand binding in
mediating receptor function.23
This study has also reported the production of several
cytokines using both murine BMDCs and human PBMCs. The
results indicate that the production of IL-12, IL-1β, and IFN-γ
follows similar structural correlations to those reported for
TLR-7 and -8 activation. Structural correlations are also noted
in the enhanced production of individual cytokines (e.g., 17 and
IL-1β and 19 and IFN-γ), suggesting it may be possible to
design compounds with selectivity for particular cytokines. This
is not a new idea. Using human PBMCs, Gorden et al. showed
that TLR-8 agonists are more potent stimulators of
proinflammatory cytokines, such as TNF-α, than TLR-7
agonists (which were more potent IFN-α stimulators).24 Our
PBMC data support this hypothesis and show a similar
correlation between TLR-8 activity and TNF-α production.
Moreover, this correlation is not apparent in the IFN-α values.
None of the compounds reported here, however, are TLR-8
selective, so a more detailed analysis of this structure−function
relationship is not possible. Nevertheless, the data reported
here suggest that the addition of TLR-8 activity generally
enhances the cytokine production of mixed agonists in both
murine BMDCs and human PBMCs. Given the prior work on
murine TLR-8 function,14−17 the ability of murine BMDCs to
register a TLR-8 dependent response in cytokine production is
noteworthy. Although beyond the scope of this study, the
results suggest that a more complete evaluation of the role
TLR-7 and -8 play in mediating the production of cytokines in
mouse models is warranted. This is especially true if mouse
models continue to be used in the design and development of
immune modifying agents and as models for evaluating
immunotherapies.
Figure 3. Human PBMC cytokine induction derived from three
donors. Average values are reported.
producing TNF-α in PBMCs than the other compounds.
Although resiquimod is also potent in producing TNF-α in
BMDCs (as noted above), the trends observed in the remaining
compounds (shown in Figure 3) are not as pronounced using
the murine cell line. This may be due in part to differences in
the function of murine and human TLR-7 and -8 receptors or
to inherent differences in the response of TNF-α to immune
modifying agents in the human and mouse.
EXPERIMENTALS
■
General Experimental Conditions. All solvents were used as
received from commercial vendors, with the exception of THF, which
was purified by passage through two alumina columns in a solvent
purification system (J. C. Meyer). All chemicals were purchased from
Sigma-Aldrich, TCI America, Combi-Blocks (boronic acid), and Strem
or were synthesized using the cited literature protocol. Compounds 1,7
2,7 12,9 16,9 1-amino-2-methylpropan-2-ol,25 1,1,1-trimethoxyhex-
a n e , 2 6 ( 2 , 2 , 2 - t r i m e t h o x y e t h y l ) b e n z e n e , 2 7 2 - ( t e r t -
butyldiphenylsilanyloxy)ethylamine,28 and 2-(phenylmethoxy)-
ethanamine29 were prepared according to the respective literature
procedure. Purity (≥95%) of all final compounds was confirmed by
reverse-phase HPLC using a mobile phase of 40% MeCN/60% 25 mM
NH4OAc buffer adjusted to pH 4−5. All reactions were conducted
under an atmosphere of N2 or Ar. Thin layer chromatography (TLC)
was performed on 0.25 mm hard-layer silica G plates. Developed plates
were visualized with a hand-held UV lamp or iodine chamber, 10%
CONCLUSION
■
An efficient synthesis for obtaining highly substituted
imidazoquinolines has been described and applied to evaluate
the SAR of a series of N1- and C2-modifed C7-
methoxycarbonyl analogues with selective TLR-7 and -8 agonist
activity and potent cytokine induction. The results show that
TLR-7 and -8 activity directly correlates with C2-alkyl chain
length, with peak activity occurring for the butyl (TLR-7) and
pentyl (TLR-8) derivatives. Only the C2-butyl and -pentyl
analogues are TLR-8 active, however, indicating that this
receptor has much more stringent requirements on binding.
TLR-8 activity is also sensitive to N1-modifications and
dramatically decreases with benzylation or methylation of the
hydroxyl group of the alkyl chain. This suggests that TLR-8
may be specific for a proton donating group at this position.
Once again, TLR-7 activity is more tolerant of substitution at
1
sulfuric acid or 10% PMA solution. H and 13C NMR spectra were
recorded on a Varian 600 MHz or a Varian 400 MHz spectrometer in
the noted solvent. Peaks are reported as chemical shift (multiplicity, J
couplings in Hz, number of protons). High resolution mass spectra
E
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were obtained on an Agilent TOF II TOF/MS instrument equipped
with either an ESI or APCI interface.
step without further purification: Rf = 0.35 (10:90, MeOH/CH2Cl2);
1H NMR (CD3OD, 600 MHz) δ 3.78 (s, 2H), 2.57 (t, J = 7.8 Hz, 2H),
General Procedure for the Synthesis of Imidazole 8. To a
suspension of aminomalononitrile p-toluenesulfonate (4.0 mmol, 1.0
equiv) in 30 mL of THF at 25 °C was added NEt3 (4.8 mmol, 1.2
equiv) in one portion. The mixture was stirred for 30 min, by which
time the mixture turned into a homogeneous solution. To this solution
was added orthoester 6 (4.8 mmol, 1.2 equiv), and the solution was
heated at reflux for 3 h before being cooled to 25 °C. NEt3 (4.8 mmol,
1.2 equiv) and primary amine 7 (4.8 mmol, 1.2 equiv) were added
sequentially, and the mixture was stirred at 25 °C for 15 h. Solvent was
removed in vacuo and the crude residue was redissolved in CH2Cl2
(100 mL) and washed with saturated Na2CO3 solution (25 mL). The
aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined
organic fractions were washed with brine, dried (MgSO4), and
concentrated in vacuo. The crude residue was purified by flash column
chromatography on silica gel.
General Procedure for the Synthesis of Imidazole 9. To a
solution of 8 (2.5 mmol, 1 equiv) in CH2I2 (5 mL) at 80 °C was added
a solution of isoamylnitrite (10.2 mmol, 4 equiv) in CHCl3 (5 mL)
over 20 min. The mixture was heated for an additional 30 min and
allowed to cool to 25 °C. The mixture was concentrated in vacuo, and
the crude residue was purified by silica gel column chromatography.
General Procedure for the Synthesis of Imidazoquinolines
13−24. Pd(OAc)2 (0.05 mmol, 0.05 equiv) and PPh3 (0.1 mmol, 0.1
equiv) were placed in a round-bottom flask and purged with argon for
10 min before DME (4 mL) was added. The resulting suspension was
stirred at 25 °C for 5 min, and 9 (1.0 mmol, 1.0 equiv), 2-amino-4-
methoxycarbonylphenylboronic acid hydrochloride salt (1.5 mmol, 1.5
equiv), and 1.5 M Na2CO3 aqueous solution (3 mmol, 3.0 equiv) were
added sequentially. The mixture was heated to 100 °C under argon for
3 h. TLC and MS analysis indicated complete conversion. The mixture
was then cooled to 25 °C and diluted with EtOAc (50 mL) and H2O
(10 mL). The aqueous layer was extracted with EtOAc (3 × 15 mL).
The combined organic layer was washed with saturated NaCl aqueous
solution (20 mL), dried over MgSO4, and concentrated in vacuo. The
resulting brown oil was loaded onto a short pad of silica gel, followed
by flashing with CH2Cl2 (30 mL). The filtrate was concentrated in
vacuo to afford the crude 11 which was used without further
purification in the next step.
1.47 (hex, J = 6.6 Hz, 2H), 1.23 (s, 6H), 0.97 (t, J = 7.8 Hz, 3H).
LRMS (ESI+): calcd C11H19N4O [M + H]+ 223.1, found 223.1.
5-Amino-1-(2-hydroxy-2-methylpropyl)-2-pentyl-1H-imida-
zole-4-carbonitrile (8f). The title compound was prepared according
to the general procedure using 1,1,1-trimethoxyhexane and 1-amino-2-
methylpropan-2-ol as an off white solid in 85% yield: Rf = 0.3 (10:90,
MeOH/CH2Cl2); 1H NMR (CD3OD, 600 MHz) δ 3.79 (s, 2H), 3.00
(t, J = 7.8 Hz, 2H), 1.69 (p, J = 7.2 Hz, 2H), 1.38−1.35 (m, 4H), 1.23
(s, 6H), 0.92 (t, J = 7.2 Hz, 3H); 13C NMR (CD3OD, 150 MHz) δ
150.8, 145.8, 117.8, 91.7, 72.8, 54.5, 32.7, 28.3, 27.6, 23.6, 14.5. HRMS
(ESI+): calcd C13H23N4O [M + H]+ 251.1872, found 251.1872 (error
0 ppm).
5-Amino-2-benzyl-1-(2-hydroxy-2-methylpropyl)-1H-imida-
zole-4-carbonitrile (8g). The title compound was prepared
according to the general procedure using (2,2,2-trimethoxyethyl)-
benzene and 1-amino-2-methylpropan-2-ol as a light yellow solid in
65% yield: Rf = 0.35 (10:90, MeOH/CH2Cl2); 1H NMR (CDCl3, 600
MHz) δ 7.31 (t, J = 7.2 Hz, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.12 (d, J =
7.2 Hz, 2H), 4.82 (br s, 2H), 3.59, (s, 2H), 3.39 (s, 2H), 2.52 (br s,
1H), 1.22 (s, 6H); 13C NMR (CDCl3, 150 MHz) δ 148.5, 142.5,
136.0, 128.9, 128.1, 127.2, 116.4, 92.4, 72.9, 53.2, 34.3, 27.7. HRMS
(ESI+): calcd C15H19N4O [M + H]+ 271.1559, found 271.1563 (error
1.5 ppm).
5-Amino-2-butyl-1-(2-hydroxypropyl)-1H-imidazole-4-car-
bonitrile (8h). The title compound was prepared according to the
general procedure using 1,1,1-trimethoxypentane and 1-amino-2-
1
methylpropanol as a beige solid in 68% yield: Rf = 0.8 (EtOAc); H
NMR (CDCl3, 400 Hz) δ 4.45 (s, 2H), 4.23−4.14 (m, 1H), 3.78 (dd, J
= 15.0, 2.54 Hz, 1H), 3.67 (dd, J = 15.0, 9.1 Hz, 1H), 3.45 (s, 1H),
2.50 (dt, J = 7.2, 2.7 Hz, 2H), 1.73−1.63 (m, 2H), 1.40 (hex, J = 7.4
Hz, 2H), 1.33 (d, J = 6.3 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H); 13C NMR
(CDCl3, 100 MHz): δ 148.3, 143.3, 118.0, 88.4, 65.1, 49.2, 28.5, 25.9,
21.8, 20.9, 13.8. HRMS (APCI+): calcd C11H19N4O [M + H]+
223.1553, found 223.1557 (error 1.5 ppm).
(S)-5-Amino-2-butyl-1-(2-hydroxypropyl)-1H-imidazole-4-
carbonitrile (8i). The title compound was prepared according to the
general procedure using 1,1,1-trimethoxypentane and (S)-1-amino-2-
methylpropanol as a beige solid in 80% yield. The analytical data
matched those of racemic 8h.
To a round-bottom flask containing 11 (0.5 mmol, 1.0 equiv) was
added 4 N HCl in dioxane (8 mmol, 16 equiv). The mixture was
heated at reflux for 4 h and then cooled to 25 °C. The mixture was
concentrated in vacuo and partitioned between 10% MeOH in EtOAc
(50 mL) and saturated NaHCO3 solution (10 mL). The aqueous layer
was extracted with 10% MeOH in EtOAc (3 × 10 mL). The combined
organic layers were washed with brine, dried over MgSO4, and
concentrated in vacuo. The crude residue was purified by flash column
chromatography on silica gel.
(R)-5-Amino-2-butyl-1-(2-hydroxypropyl)-1H-imidazole-4-
carbonitrile (8j). The title compound was prepared according to the
general procedure using 1,1,1-trimethoxypentane and (R)-1-amino-2-
methylpropanol as a beige solid in 73% yield. The analytical data
matched those of racemic 8h.
5-Amino-2-butyl-1-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-
1H-imidazole-4-carbonitrile (8k). The title compound was
prepared according to the general procedure using 1,1,1-trimethox-
ypentane and 2-(tert-butyldiphenylsilanyloxy)ethylamine and was
carried on directly to the next step.
5-Amino-1-(2-(benzyloxy)ethyl)-2-butyl-1H-imidazole-4-car-
bonitrile (8l). The title compound was prepared according to the
general procedure using 1,1,1-trimethoxypentane and 2-
(phenylmethoxy)ethanamine as a beige solid in 72% yield: Rf = 0.25
(20:80, EtOAc/hexanes); 1H NMR (CDCl3, 400 Hz) δ 7.39−7.29 (m,
3H), 7.24−7.19 (m, 2H), 4.51 (s, 2H), 4.38 (s, 2H), 3.95 (t, J = 4.7
Hz, 2H), 3.70 (t, J = 4.7 Hz, 2H), 2.49 (t, J = 7.6 Hz, 2H), 1.86 (p, J =
7.8 Hz, 2H), 1.37 (hex, J = 7.43 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13C
NMR (CDCl3, 100 MHz): δ 147.2, 143.7, 136.6, 128.7, 128.3, 127.7,
116.4, 93.1, 73.8, 69.7, 43.4, 29.2, 26.8, 22.4, 13.7. HRMS (APCI+):
calcd C17H23N4O [M + H]+ 299.1886, found 299.1874 (error 2.5
ppm).
5-Amino-1-(2-hydroxy-2-methylpropyl)-2-methyl-1H-imida-
zole-4-carbonitrile (8b). The title compound was prepared
according to the general procedure using 1,1,1-trimethoxyethane and
1-amino-2-methylpropan-2-ol as an off white solid in 69% yield: Rf =
0.25 (10:90, MeOH/CH2Cl2); 1H NMR (CD3OD), 600 MHz) δ 3.78
(s, 2H), 2.27 (s, 3H), 1.24 (s, 6H); 13C NMR (CD3OD), 150 MHz) δ
150.9, 143.2, 117.7, 90.6, 72.8, 55.0, 27.5, 14.0. HRMS (ESI+): calcd
C9H15N4O [M + H]+ 195.1240, found 195.1237 (error 1.5 ppm).
5-Amino-2-ethyl-1-(2-hydroxy-2-methylpropyl)-1H-imida-
zole-4-carbonitrile (8c). The title compound was prepared
according to the general procedure using 1,1,1-trimethoxypropane
and 1-amino-2-methylpropan-2-ol as an off white solid in 75% yield: Rf
1
= 0.30 (10:90, MeOH/CH2Cl2); H NMR (CD3OD, 400 MHz) δ
3.79 (s, 2H), 2.35 (q, J = 7.6 Hz, 2H), 1.27−1.23 (m, 9H); 13C NMR
(CDCl3, 100 MHz) δ 150.9, 147.8, 117.8, 91.0, 72.8, 54.5, 27.5, 21.5,
11.9. HRMS (ESI+): calcd C10H17N4O [M + H]+ 209.1397, found
209.1405 (error 3.8 ppm).
5-Amino-2-butyl-1-(2-methoxyethyl)-1H-imidazole-4-car-
bonitrile (8m). The title compound was prepared according to the
general procedure using 1,1,1-trimethoxypentane and 2-methoxye-
thanamine as a beige solid in 72% yield: Rf = 0.24 (80:20, EtOAc/
1
hexanes); H NMR (CDCl3, 400 Hz) δ 4.41 (s, 2H), 3.93 (t, J = 4.8
5-Amino-1-(2-hydroxy-2-methylpropyl)-2-propyl-1H-imida-
zole-4-carbonitrile (8d). The title compound was prepared
according to the general procedure using 1,1,1-trimethoxybutane and
1-amino-2-methylpropan-2-ol and was carried on directly to the next
Hz, 2H), 3.61 (t, J = 4.8 Hz, 2H), 3.37 (s, 3H), 2.53 (t, J = 7.7 Hz,
2H), 1.70 (p, J = 7.6 Hz, 2H), 1.41 (hex, J = 7.4 Hz, 2H), 0.95 (t, J =
7.43 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 147.4, 143.7, 116.4,
F
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Journal of Medicinal Chemistry
Article
93.1, 72.5, 59.3, 43.4, 29.3, 26.9, 22.4, 13.8. HRMS (APCI+): calcd
C11H19N4O [M + H]+ 223.1553, found 223.1582 (error 3.9 ppm).
1-(2-Hydroxy-2-methylpropyl)-5-iodo-2-methyl-1H-imida-
zole-4-carbonitrile (9b). The title compound was prepared
according to the general procedure using 8b as a yellow foam in
56% yield: Rf = 0.50 (50:50, EtOAc/hexanes); 1H NMR (CDCl3, 600
MHz) δ 3.94 (s, 2H), 2.57 (s, 3H), 1.33 (s, 6H); 13C NMR (CDCl3,
150 MHz) δ 151.7, 120.5, 114.9, 83.5, 71.8, 57.1, 28.4, 15.6. HRMS
(APCI+): calcd C9H13IN3O [M + H]+ 306.0098, found 306.0090
(error 2.6 ppm).
115.0, 81.2, 62.0, 49.0, 29.1, 27.7, 26.7, 22.4, 19.0, 13.8. HRMS (APCI
+): calcd C26H33ISiN3O [M + H]+ 558.1432, found 558.1432 (error
0.02 ppm).
1-(2-(Benzyloxy)ethyl)-2-butyl-5-iodo-1H-imidazole-4-car-
bonitrile (9l). The title compound was prepared according to the
general procedure using 8l as a black solid in 30% yield: Rf = 0.35
1
(30:70, EtOAc/hexanes); H NMR (CDCl3, 400 MHz) δ 7.40−7.12
(m, 5H), 4.46 (t, 2H), 4.13 (t, J = 5.2 Hz, 2H), 3.68 (t, J = 5.2 Hz,
2H), 2.78 (t, J = 7.6 Hz, 2H), 1.72 (p, J = 7.8 Hz, 2H), 1.37 (hex, J =
7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ
154.3, 140.0, 128.5, 128.1, 127.6, 120.9, 114.9, 80.9, 73.4, 67.7, 47.5,
29.2, 27.5, 22.3, 13.7. HRMS (APCI+): calcd C17H21IN3O [M + H]+
410.0724, found 410.0723 (error 0.2 ppm).
2-Ethyl-1-(2-hydroxy-2-methylpropyl)-5-iodo-1H-imidazole-
4-carbonitrile (9c). The title compound was prepared according to
the general procedure using 8c as a yellow foam in 56% yield: Rf = 0.55
1
(50:50, EtOAc/hexanes); H NMR (CD3OD, 400 MHz) δ 4.05 (s,
2-Butyl-5-iodo-1-(2-methoxyethyl)-1H-imidazole-4-carboni-
trile (9m). The title compound was prepared according to the general
procedure using 8m as an amber solid in 29% yield (two steps): Rf =
0.52 (50:50, EtOAc/hexanes); 1H NMR (CDCl3, 400 MHz) δ 4.12 (t,
J = 5.3 Hz, 2H), 3.60 (t, J = 5.3 Hz, 2H), 3.31 (s, 3H), 2.79 (t, J = 2.8
Hz, 2H), 1.74 (p, J = 7.6 Hz, 2H), 1.42 (hex, J = 7.4 Hz, 2H), 0.96 (t, J
= 7.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 154.3, 120.9, 114.9,
81.0, 70.7, 59.2, 47.5, 29.2, 27.5, 22.3, 13.8. HRMS (APCI+): calcd
C11H17IN3O [M + H]+ 334.0411, found 334.0416 (error 1.6 ppm).
4-Amino-1-(2-hydroxyl-2-methylpropyl)-7-methoxycarbon-
yl-2-methyl-1H-imidazo[4,5-c]quinoline (13). The title com-
pound was prepared according to the general procedure using 9b as
a off white solid in 75% yield over two steps: Rf = 0.50 (10:90, MeOH/
2H), 2.98 (q, J = 7.6 Hz, 2H), 1.25−1.30 (m, 9H); 13C NMR
(CD3OD, 100 MHz) δ 158.1, 121.2, 116.3, 87.7, 72.1, 58.3, 28.5, 23.0,
12.1. HRMS (APCI+): calcd C10H15IN3O [M + H]+ 320.0254, found
320.0268 (error 4.4 ppm).
1-(2-Hydroxy-2-methylpropyl)-5-iodo-2-propyl-1H-imida-
zole-4-carbonitrile (9d). The title compound was prepared
according to the general procedure using 8d as a yellow foam in
1
56% yield: Rf = 0.55 (50:50, EtOAc/hexanes); H NMR (CD3OD,
400 MHz) δ 4.85 (s, 2H), 2.93 (t, J = 7.6 Hz, 2H), 1.74 (hex, J = 7.6
Hz, 2H), 1.25 (s, 6H), 0.98 (t, J = 7.6 Hz, 3H); 13C NMR (CD3OD,
100 MHz) δ 157.0, 121.2, 116.3, 87.6, 72.1, 58.3, 31.5, 28.5, 22.1, 14.2.
HRMS (APCI+): calcd C11H17IN3O [M + H]+ 334.0411, found
334.0408 (error 0.9 ppm).
1
CH2Cl2); H NMR (CD3OD, 400 MHz) δ 8.37 (d, J = 8.4 Hz, 1H),
1-(2-Hydroxy-2-methylpropyl)-5-iodo-2-pentyl-1H-imida-
zole-4-carbonitrile (9f). The title compound was prepared according
to the general procedure using 8f as a yellow foam in 66% yield: Rf =
0.45 (50:50, EtOAc/hexanes); 1H NMR (CDCl3, 600 MHz) δ 3.96 (s,
2H), 2.89 (t, J = 7.2 Hz, 2H), 2.00 (br s, 1H), 1.71 (p, J = 7.8 Hz, 2H),
1.31 (s, 6H), 1.20−1.35 (m, 4H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3, 150 MHz) δ 155.4, 120.9, 115.1, 83.3, 71.7, 56.6, 31.4, 28.6,
28.4, 27.3, 22.3, 13.9. HRMS (ESI+): calcd C13H21IN3O [M + H]+
362.0729, found 362.0731 (error 0.5 ppm).
8.33 (d, J = 2.0 Hz, 1H), 7.86 (dd, J = 8.8, 2.0 Hz, 1H), 4.65 (br s,
2H), 3.96 (s, 3H), 2.80 (s, 3H), 1.30 (br s, 6H); 13C NMR (DMSO-d6,
150 MHz) δ 166.6, 152.8, 152.4, 144.2, 133.1, 127.5, 127.4, 126.6,
121.6, 119.7, 118.7, 70.9, 55.0, 52.0, 27.6, 14.7. HRMS (ESI+): calcd
C17H21N4O3 [M + H]+ 329.1608, found 329.1602 (error 1.8 ppm).
4-Amino-2-ethyl-1-(2-hydroxyl-2-methylpropyl)-7-methoxy-
carbonyl-1H-imidazo[4,5-c]quinoline (14). The title compound
was prepared according to the general procedure using 9c as a white
solid in 40% yield over two steps: Rf = 0.50 (10:90, MeOH/CH2Cl2);
1H NMR (CD3OD, 600 MHz) δ 8.38 (d, J = 9.0 Hz, 1H), 8.15 (d, J =
1.8 Hz, 1H), 7.70 (dd, J = 9.0, 1.8 Hz, 1H), 6.62 (br s, 2H), 4.79 (br s,
2H), 3.88 (s, 3H), 3.05 (q, J = 7.8 Hz, 2H), 1.35 (t, J = 7.8 Hz, 3H),
1.23 (br s, 6H); 13C NMR (DMSO-d6, 150 MHz) δ 166.6, 157.2,
152.4, 144.2, 133.1, 121.7, 119.8, 118.8, 70.8, 54.6, 52.1, 27.0, 20.6,
12.1. HRMS (ESI+): calcd C18H23N4O3 [M + H]+ 343.1765, found
343.1764 (error 0.3 ppm).
4-Amino-1-(2-hydroxyl-2-methylpropyl)-7-methoxycarbon-
yl-2-propyl-1H-imidazo[4,5-c]quinoline (15). The title compound
was prepared according to the general procedure using 9d as a white
solid in 34% yield over two steps: Rf = 0.50 (10:90, MeOH/CH2Cl2);
1H NMR (CD3OD, 600 MHz) δ 8.34 (d, J = 8.4 Hz, 1H), 8.32 (s,
1H), 7.84 (d, J = 8.4 Hz, 1H), 4.59 (br s, 2H), 3.95 (s, 3H), 3.10 (t, J =
7.8 Hz, 2H), 1.94 (hex, J = 7.8 Hz, 2H), 1.28 (br s, 6H), 1.09 (t, J =
7.2 Hz, 3H); 13C NMR (DMSO-d6, 150 MHz) δ 168.9, 158.7, 153.7,
145.1, 135.6, 129.5, 128.7, 128.5, 122,9, 122.6, 120.1, 72.7, 56.4, 52.9,
30.9, 28.0, 22.4, 14.4. HRMS (ESI+): calcd C19H25N4O3 [M + H]+
357.1921, found 357.1908 (error 3.6 ppm).
4-Amino-1-(2-hydroxyl-2-methylpropyl)-7-methoxy carbon-
yl-2-pentyl-1H-imidazo[4,5-c]quinoline (17). The title compound
was prepared according to the general procedure using 9f as a white
solid in 42% yield over two steps: Rf = 0.45 (10:90, MeOH/CH2Cl2);
1H NMR (DMF-d7, 600 MHz) δ 8.53 (d, J = 9.0 Hz, 1H), 8.31 (d, J =
1.8 Hz, 1H), 7.78 (dd, J = 8.4, 1.8 Hz, 1H), 6.69 (br s, 2H), 5.01 (s,
1H), 4.67 (br s, 2H), 3.96 (s, 3H), 3.14 (t, J = 7.8 Hz, 2H), 1.91 (p, J =
7.2 Hz, 2H), 1.42−1.46 (m, 4H), 1.31 (br s, 6H), 0.92 (t, J = 7.2 Hz,
3H); 13C NMR (DMF-d7, 150 MHz) δ 167.8, 157.7, 153.6, 145.5,
134.4, 129.0, 128.8, 128.0, 122.8, 121.0, 120.1, 72.0, 55.8, 52.6, 32.4,
28.4, 28.3, 28.2, 23.2, 14.5. HRMS (ESI+): calcd C21H29N4O3 [M+H]+
385.2234, found 385.2234 (error 0 ppm).
2-Benzyl-1-(2-hydroxy-2-methylpropyl)-5-iodo-1H-imida-
zole-4-carbonitrile (9g). The title compound was prepared
according to the general procedure using 8g as a light yellow foam
1
in 70% yield: Rf = 0.55 (50:50, EtOAc/hexanes); H NMR (CDCl3,
600 MHz) δ 7.31 (t, J = 7.2 Hz, 2H), 7.26 (t, J = 6.6 Hz, 1H), 7.16 (d,
J = 7.2 Hz, 2H), 4.46 (s, 2H), 3.84 (s, 2H), 1.62 (br s, 1H), 1.32 (s,
6H); 13C NMR (CDCl3, 150 MHz) δ 153.5, 135.9, 128.9, 128.6,
127.1, 114.9, 84.2, 72.1, 56.5, 35.0, 28.7. HRMS (ESI+): calcd
C15H17IN3O [M + H]+ 382.0416, found 382.0409 (error 1.8 ppm).
2-Butyl-1-(2-hydroxypropyl)-5-iodo-1H-imidazole-4-carbon-
itrile (9h). The title compound was prepared according to the general
procedure using 8h as an amber solid in 31% yield: Rf = 0.33 (50:50,
1
EtOAc/hexanes); H NMR (CDCl3, 400 Hz) δ 4.24−4.14 (m, 1H),
3.94−3.82 (m, 2H), 2.88−2.72 (m, 2H), 2.19 (d, J = 3.7 Hz, 1H), 1.73
(p, J = 7.43 Hz, 2H), 1.41 (hex, J = 7.4 Hz, 2H), 1.35 (d, J = 6.2 Hz,
3H), 0.95 (t, J = 7.4 Hz, 3H); 13 C NMR (400 MHz, CDCl3) δ 154.5,
120.6, 114.8, 81.6, 66.8, 54.4, 29.3, 27.7, 22.3, 21.0, 13.7. HRMS
(APCI+): calcd C11H17IN3O [M + H]+ 334.0411, found 334.0406
(error 1.45 ppm).
(S)-2-Butyl-1-(2-hydroxypropyl)-5-iodo-1H-imidazole-4-car-
bonitrile (9i). The title compound was prepared according to the
general procedure using 8i as an amber solid in 21% yield. The
analytical data matched those of racemic 8h.
(R)-2-Butyl-1-(2-hydroxypropyl)-5-iodo-1H-imidazole-4-car-
bonitrile (9j). The title compound was prepared according to the
general procedure using 8j as an amber solid in 33% yield. The
analytical data matched those of racemic 8h.
2-Butyl-1-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-5-iodo-1H-
imidazole-4-carbonitrile (9k). The title compound was prepared
according to the general procedure using 8k as a black solid in 21%
1
yield over two steps. Rf = 0.48 (30:70, EtOAc/hexanes); H NMR
(CDCl3, 400 MHz) δ 7.50−7.35 (m, 10H), 4.09 (t, J = 5.4 Hz, 2H),
3.85 (t, J = 5.4 Hz, 2H), 2.81−2.74 (m, 2H), 1.70 (p, J = 7.4 Hz, 2H),
1.38 (hex, J = 7.43 Hz, 2H), 1.01 (s, 9H), 0.94 (t, J = 7.4 Hz, 3H); 13C
NMR (CDCl3, 100 MHz) δ 154.1, 135.4, 132.1, 130.1, 128.0, 120.9,
4-Amino-2-benzyl-1-(2-hydroxyl-2-methylpropyl)-7-me-
thoxycarbonyl-1H-imidazo[4,5-c]quinoline (18). The title com-
pound was prepared according to the general procedure using 9g as an
off white solid in 45% yield over two steps: Rf = 0.50 (10:90, MeOH/
G
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Journal of Medicinal Chemistry
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1
CH2Cl2); H NMR (DMF-d7, 600 MHz) δ 8.49 (d, J = 9.0 Hz, 1H),
TLR7/8-NF-κB Reporter Assay. Human embryonic kidney
(HEK) cells that were stably transfected with human TLR-7 or
TLR-8 and an NF-κB-responsive secreted embryonic alkaline
phosphatase (SEAP) gene (HEK-TLR-7 and -8) were purchased
from InvivoGen (San Diego, CA). The procedure used to measure
TLR7 or TLR8 agonist activity was conducted as described by Hood
et al.1 HEK-TLR7/8 cells were stimulated with 30 μM compound in a
96-well plate in DMEM containing 10% FBS and 0.01% Normocin
(InvivoGen) for 24 h. Then 20 μL of the supernatant from each well
was incubated with Quanti-blue substrate solution (InvivoGen) at 37
°C for 1 h and absorbance was read at 650 nm using a Synergy plate
reader (Biotek, Winooski, VT).
Measurement of Proimflammatory Cytokines. Bone marrow
derived dendritic cells (BMDC) were generated by isolating a single
cell suspension of marrow from the femur of C57BL/6 mice (6−8
weeks of age). Red blood cells were lysed with 0.83% NH4Cl, 0.1%
KHCO3, and 0.009% EDTA. Five million cells were seeded in each of
well of a six-well plate in complete RPMI medium (Invitrogen,Grand
Island, NY) and supplemented with mouse 20 ng/mL granulocyte-
macrophage colony stimulating factor (PeproTech, Rocky Hill, NJ).
Six days after culture, BMDCs were stimulated with 30 μM compound
for 3 days. An amount of 25 μL of supernatant was then removed and
assayed for TNF-α, IL-12p40, IL-1β, and IL-10 using a flow cytometric
bead array according to the manufacturers’ instructions (BD
Bioscience, San Jose,CA). Peripheral blood mononuclear cells
(PBMCs) were isolated from three independent blood donor samples
using the Ficoll method and plated in triplicate at a concentration of 5
× 105 cells in a 24-well plate. The cells were stimulated at a compound
concentration of 22.5 μM and incubated for 24 h. An amount of 50 μL
of medium was isolated and analyzed for TNF-α and IFN-γ by bead
array (as described above) or IFN-α by ELISA (eBioscience, San
Diego, CA). Flow cytometry was performed on a FACSCanto II (BD
Bioscience), and data were analyzed using Flowjo software (Tree Star,
Inc., Ashland, OR).
8.30 (s, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.33−7.35 (m, 4H), 7.25−7.27
(m, 1H), 6.78 (br s, 2H), 5.28 (br s, 1H), 4.65 (br s, 4H), 3.95 (s, 3H),
1.33 (br s, 6H); 13C NMR (DMF-d7, 150 MHz) δ 167.8, 155.8, 153.8,
145.8, 138.7, 134.7, 129.7, 129.5, 129.2, 129.1, 128.3, 127.5, 122.7,
121.1, 120.1, 72.1, 56.2, 52.6, 34.9, 28.1. HRMS (ESI+): calcd
C23H25N4O3 [M + H]+ 405.1927, found 405.1935 (error 2.0 ppm).
)-4-Amino-2-butyl-1-(2-hydroxypropyl)-7-methoxycar-
bonyl-1H-imidazo[4,5-c]quinoline (19). The title compound was
prepared according to the general procedure using 9h as a white solid
(
1
in 51% yield over two steps: Rf = 0.4 (10:90, MeOH/EtOAc); H
NMR (DMF-d7, 400 MHz) δ 8.31 (d, J = 1.61 Hz, 1H), 8.24 (d, J =
8.7 Hz, 1H), 7.80 (dd, J = 8.5, 1.84 Hz, 1H), 6.68 (s, 2H), 5.29 (s,
1H), 4.70 (dd, J = 15.2, 3.22 Hz, 1H), 4.46 (dd, J = 14.94, 9.49 Hz,
1H), 4.32−4.21 (m, 1H), 3.95 (s, 3H), 3.12−3.02 (m, 2H), 1.90 (p, J
= 7.8 Hz, 2H), 1.52 (hex, J = 7.3 Hz, 2H), 1.39 (d, J = 6.2 Hz, 3H),
0.99 (t, J = 7.3 Hz, 3H); 13C NMR (DMF-d7, 100 MHz) δ 167.1,
156.1, 153.1, 144.5, 132.8, 128.4, 128.3, 127.6, 121.2, 121.1, 119.0,
66.3, 52.8, 52.0, 29.7, 27.3, 22.6, 20.8, 13.8. HRMS (APCI+): calcd
C19H25N4O3 [M + H]+ 357.1921, found 357.1926 (error 1.4 ppm).
(S)-4-Amino-2-butyl-1-(2-hydroxypropyl)-7-methoxycar-
bonyl-1H-imidazo[4,5-c]quinoline (20). The title compound was
prepared according to the general procedure using 9i as a white solid
in 64% yield over two steps. The analytical data matched those of
racemic 19. The absolute stereochemistry was confirmed using
Mosher ester analysis following the Hoye protocol.30 The use of
(R)-MTPA resulted in a 19F NMR peak at −74.5.
(R)-4-Amino-2-butyl-1-(2-hydroxypropyl)-7-methoxycar-
bonyl-1H-imidazo [4,5-c]quinoline (21). The title compound was
prepared according to the general procedure using 9j as a white solid
in 50% yield over two steps. The analytical data matched those of
racemic 19. The absolute stereochemistry was confirmed using
Mosher ester analysis following the Hoye protocol.29 The use of
(R)-MTPA resulted in a 19F NMR peak at −74.2.
4-Amino-2-butyl-1-(2-hydroxyethyl)-7-methoxycarbonyl-
1H-imidazo[4,5-c]quinoline (22). The title compound was
prepared according to the general procedure using 9k as a white
solid in 23% yield over two steps: Rf = 0.2 (10:90, MeOH/CH2Cl2);
1H NMR (DMF-d7, 400 MHz) δ 8.37−8.27 (m, 2H), 7.82 (dd, J =
AUTHOR INFORMATION
Corresponding Author
*Phone: 612-626-2601. E-mail: ferguson@umn.edu.
■
8.61, 1.76 Hz, 1H), 6.95 (s, 2H), 5.28 (t, J = 5.2 Hz, 1H), 4.77 (t, J =
5.2 Hz, 2H), 4.10−4.05 (m, 2H), 3.96 (s, 3H), 3.07 (t, J = 7.8 Hz,
2H), 1.90 (p, J = 7.63 Hz, 2H), 1.52 (hex, J = 7.6 Hz, 2H), 0.99 (t, J =
7.4 Hz, 3H); 13C NMR (DMF-d7, 100 MHz) δ 167.8, 157.0, 153.7,
133.7, 129.2, 128.6, 122.1, 122.0, 119.5, 61.6, 52.8, 48.9, 27.9, 23.4,
14.5 (peak missing at ∼144). HRMS (APCI+): calcd C18H23N4O3 [M
+ H]+ 343.1765, found 343.1754 (error 3.1 ppm).
4-Amino-1-(2-(benzyloxy)ethyl)-2-butyl-7-methoxycarbonyl
1H-imidazo[4,5-c]quinoline (23). The title compound was
prepared according to the general procedure using 9l as a white
solid in 27% yield over two steps: Rf = 0.50 (10:90, MeOH/CH2Cl2);
1H NMR (CDCl3, 400 Hz) δ 8.53 (s, 1H), 7.95−7.85 (m, 2H), 7.26−
7.09 (m, 5H), 5.48 (s, 2H), 4.70 (t, J = 5.6 Hz, 2H), 4.49 (s, 2H), 3.96
(s, 3H), 3.94 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 7.8 Hz, 2H), 1.86 (p, J =
7.83 Hz, 2H), 1.49 (hex, J = 7.43 Hz, 2H), 0.98 (t, J = 7.43 Hz, 3H);
13C NMR (CDCl3, 100 MHz): δ 167.3, 155.4, 151.6, 144.0, 137.0,
132.7, 129.3, 128.4, 127.9, 127.6, 122.1, 119.3, 118.5, 73.5, 67.9, 52.2,
45.6, 29.9, 27.2, 22.6, 13.8. HRMS (APCI+): calcd C25H29N4O3 [M +
H]+ 433.2234, found 433.2235 (error 0.14 ppm).
Notes
The authors declare no competing financial interest.
⊥J.R.O.: Deceased January 21, 2013.
ACKNOWLEDGMENTS
■
The authors thank Dr. Chris Pennell for reading the manuscript
and providing helpful comments and suggestions with regard to
immunology and financial support from the Minnesota Futures
Grant Program.
ABBREVIATIONS USED
■
IFN, interferon; IL, interleukin; TNF, tumor necrosis factor;
NF-κB, nuclear factor κB; MyD88, myeloid differentiation
protein 88; PBMC, peripheral blood mononuclear cell; BMDC,
bone marrow derived dendritic cell; TLR, Toll-like receptor;
CD8, cluster of differentiation 8; CD4, cluster of differentiation
4; APC, antigen presenting cell
4-Amino-2-butyl-1-(2-methoxyethyl)-7-methoxycarbonyl-
1H-imidazo[4,5-c]quinoline (24). The title compound was
prepared according to the general procedure using 9m as a white
solid in 43% yield over two steps: Rf = 0.3 (10:90, MeOH/EtOAc); 1H
NMR (DMF-d7, 400 MHz) δ 8.31 (d, J = 1.76 Hz, 1H), 8.27 (d, J =
8.61 Hz, 1H), 7.80 (dd, J = 8.6, 1.7 Hz, 1H), 6.69 (s, 2H), 4.86 (t, J =
5.2 Hz, 2H), 3.96 (s, 3H), 3.91 (t, J = 5.2 Hz, 2H), 3.26 (s, 3H), 3.03
(t, J = 7.6 Hz, 2H), 1.89 (p, J = 7.6 Hz, 2H), 1.52 (hex, J = 7.6 Hz,
2H), 0.99 (t, J = 7.4 Hz, 3H); 13C NMR (DMF-d7, 100 MHz) δ 168.0,
156.5, 154.0, 145.8, 133.4, 129.5, 129.4, 128.5, 121.7, 119.7, 72.2, 59.5,
52.8, 46.4, 27.7, 23.4, 14.5. HRMS (APCI+): calcd C19H25N4O3 [M +
H]+ 357.1921, found 357.1920 (error 0.3 ppm).
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