Exquisite selectivity for human toll-like receptor 8 in substituted furo[2,3-c]quinolines

Article abstract of DOI:10.1021/jm400694d

Toll-like receptor (TLR)-8 agonists activate adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds may be promising candidate adjuvants. We synthesized and evaluated hitherto unexplored furo[2,3-c]quinolines and regioisomeric furo[3,2-c]quinolines derived via a tandem, one-pot Sonogashira coupling and intramolecular 5-endo-dig cyclization strategy in a panel of primary screens. We observed a pure TLR8-agonistic activity profile in select furo[2,3-c] quinolines, with maximal potency conferred by a C2-butyl group (EC₅₀ = 1.6 μM); shorter, longer, or substituted homologues as well as compounds bearing C1 substitutions were inactive, which was rationalized by docking studies using the recently described crystal structure of human TLR8. The best-in-class compound displayed prominent proinflammatory cytokine induction (including interleukin-12 and interleukin-18), but was bereft of interferon-α inducing properties, confirming its high selectivity for human TLR8.

Full text of DOI:10.1021/jm400694d

Article
pubs.acs.org/jmc
Exquisite Selectivity for Human Toll-Like Receptor 8 in Substituted
Furo[2,3‑c]quinolines
Hari Prasad Kokatla,† Diptesh Sil,† Subbalakshmi S. Malladi,† Rajalakshmi Balakrishna,†
Alec R. Hermanson,† Lauren M. Fox,† Xinkun Wang,‡ Anshuman Dixit,§ and Sunil A. David*,†
‡
†Department of Medicinal Chemistry and Genomics Facility, University of Kansas, Lawrence, Kansas 66047, United States
§Department of Translational Research and Technology Development, Institute of Life Sciences, Nalco Square, Bhubaneswar 751023,
India
S
* Supporting Information
ABSTRACT: Toll-like receptor (TLR)-8 agonists activate
adaptive immune responses by inducing robust production of
T helper 1-polarizing cytokines, suggesting that TLR8-active
compounds may be promising candidate adjuvants. We
synthesized and evaluated hitherto unexplored furo[2,3-
c]quinolines and regioisomeric furo[3,2-c]quinolines derived
via a tandem, one-pot Sonogashira coupling and intramolecular
5-endo-dig cyclization strategy in a panel of primary screens.
We observed a pure TLR8-agonistic activity profile in select
furo[2,3-c]quinolines, with maximal potency conferred by a
C2-butyl group (EC50 = 1.6 μM); shorter, longer, or
substituted homologues as well as compounds bearing C1
substitutions were inactive, which was rationalized by docking studies using the recently described crystal structure of human
TLR8. The best-in-class compound displayed prominent proinflammatory cytokine induction (including interleukin-12 and
interleukin-18), but was bereft of interferon-α inducing properties, confirming its high selectivity for human TLR8.
which has been associated with allergic reactions in some
subjects.7,8
INTRODUCTION
■
Vaccines have played an indispensable role in the dramatic
improvement in public health witnessed in modern times,
particularly in the prevention of mortality and morbidity
attributable to infectious diseases.1 Although the successes of
active immunization beginning with Jenner’s smallpox vaccine
in 1796 are numerous and extraordinary, our failure to develop
effective vaccines against diseases such as the human
immunodeficiency virus (HIV), tuberculosis, and malaria have
served to highlight the deficiencies and shortcomings of state-
of-the-art contemporary vaccine technology, catalyzing a
renaissance in rational vaccine design and development.2,3
A component that is being increasingly recognized as pivotal
in the design of effective vaccines is the incorporation of
appropriate immune potentiators (also termed adjuvants) along
with the antigen. Adjuvants initiate early innate immune
responses, which subsequently lead to the induction of robust
and long-lasting adaptive immune responses.4 Aluminum salts
(primarily phosphate and hydroxide), discovered by Glenny
and co-workers,5 have been the only adjuvants in clinical use
until the recent approval of 3-O-desacyl-4′-monophosphoryl
lipid A (MPL) by the FDA.6 Aluminum salts are weak
adjuvants for antibody induction, promoting a T helper 2
(Th2)-skewed, rather than a Th1, response;7,8 they are virtually
ineffective at inducing cytotoxic T lymphocyte or mucosal
immunoglobulin A (IgA) antibody responses, and they also
appear to promote the induction of IgE isotype switching,
Innate immune afferent signals activated by vaccine adjuvants
include those originating from Toll-like receptors (TLRs) as
well as retinoic acid-inducible gene 1 (RIG-I)-like receptors9
and nucleotide oligomerization domain (NOD)-like receptors
(NLRs).10,11 There are 10 functional TLRs encoded in the
human genome, which are transmembrane proteins with an
extracellular domain having leucine-rich repeats (LRR) and a
cytosolic domain called the Toll/IL-1 receptor (TIR)
domain.12 The ligands for these receptors are highly conserved
molecules such as lipopolysaccharides (LPS) (recognized by
TLR4), lipopeptides (TLR2 in combination with TLR1 or
TLR6), flagellin (TLR5), single-stranded RNA (TLR7 and
TLR8), double-stranded RNA (TLR3), CpG-motif-containing
DNA (recognized by TLR9), and profilin present on
uropathogenic bacteria (TLR11).12 TLR1, -2, -4, -5, and -6
recognize extracellular stimuli, whereas TLR3, -7, -8, and -9
function within the endolysosomal compartment.12 The
activation of TLRs by their cognate ligands leads to the
production of inflammatory cytokines and the upregulation of
major histocompatibility complex (MHC) molecules and
costimulatory signals in antigen-presenting cells (APCs) as
well as activating natural killer (NK) cells (innate immune
Received: May 9, 2013
© XXXX American Chemical Society
A
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response), which lead to the priming and amplification of T-
and B-cell effector functions (adaptive immune re-
sponses).13−16
Driven by our desire to identify pure TLR8 agonists also
capable of inducing IL-12 and IL-18 and drawing from our
earlier work in delineating structure−activity relationships
(SAR) in the furo[2,3-c]pyridines, we began exploring a variety
of fused heterocyclic core structures. Our continuing
investigations have led to the discovery of pure TLR8-agonistic
activity associated with strong interferon-γ (IFN-γ)-, IL-12-,
and IL-18-inducing activities in a series of furo[2,3-c]quinolines.
The 4-amino-furo[2,3-c]quinoline chemotype is unprecedented
in the literature, and the activity profile of this class of
compounds, examined by a range of secondary screens in
human ex vivo blood models, including transcriptomal
profiling, confirmed pure TLR8 agonism with no detectable
signatures of TLR7 activity, allowing for the first time a clear
path toward the evaluation of such compounds as potential
adjuvants for vaccines.
We have recently embarked on a systematic exploration17,18
of a variety of TLR agonists with a view toward identifying safe
and potent vaccine adjuvants. The chemotypes that we have
explored thus far include agonists of TLR2,19−21 TLR7,22−26
TLR8,27,28 NOD1,29 and C−C chemokine receptor type 1.30
Our efforts are currently focused on evaluating small-
molecule agonists of TLR8 as potential vaccine adjuvants.
TLR8 is expressed in myeloid DCs, monocytes, and monocyte-
derived dendritic cells, and the engagement of TLR8 agonists
induces a dominant proinflammatory cytokine profile including
tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), and
IL-18.31 The prominent nuclear factor-κB (NF-κB)- and c-Jun
N-terminal kinase (JNK)-mediated stimulatory effects of TLR8
agonists on APCs32,33 lead to robust Th1-type responses.34
Unlike TLR2, -4, or -7 agonists, TLR8 agonists appear unique
in their ability to markedly upregulate the production of the
Th1-polarizing cytokines TNF-α and IL-12 from neonatal
APCs,34suggesting that TLR8 agonists may be useful as
adjuvants for enhancing immune responses in newborns.
Furthermore, human T-regulatory cells (Tregs), classified
immunophenotypically as naturally occurring
(CD4+CD25+Foxp3+) or induced (CD4+CD25high), down-
regulate and suppress a broad array of immune responses,
including the nonspecific suppression of both CD4+ and CD8+
T-cells via cell−cell contact and production of immunosup-
pressive cytokines such as IL-10 and transforming growth
factor-β (TGF-β).35−41 Tregs express abundant TLR8 mRNA,
and TLR8 agonists have been shown to reverse Treg function
via a TLR8−MyD88 (myeloid differentiation factor 88)−
IRAK4 (IL-1-receptor-associated kinase 4) signaling pathway.42
The engagement and activation of TLR8 is, therefore, strongly
associated with enhanced adaptive immune responses.
RESULTS AND DISCUSSION
■
Our recent work on the 2,3-diamino-furo[2,3-c]pyridines,
which display TLR8-dependent NF-κB signaling but are
dissociated from downstream cytokine induction, led us to
consider benzologues of the furo[2,3-c]pyridines. The 4-amino-
furo[2,3-c]quinoline chemotype is hitherto not described in the
literature, and it presented a good starting point because its
scaffold closely resembles known TLR7/8-active ligands. We
envisioned that the furo[2,3-c]quinoline and its regioisomeric
core structures could be derived via one-pot Sonogashira
coupling of alkynes to either 4-iodoquinolin-3-ol (Scheme 1) or
3-iodoquinolin-4-ol (Scheme 2) followed by a tandem, tethered
nucleophile-assisted, intramolecular 5-endo-dig cyclization.50
Electrophilic iodination of commercially available 3- and 4-
Scheme 1. Syntheses of C2-Alkylfuro[2,3-c]quinoline
a
Analogues
A prerequisite for the careful evaluation of TLR8 activators as
potential adjuvants for neonatal vaccines is the availability of
pure TLR8 agonists with negligible TLR7 activity and, other
than the 2,3-diamino-furo[2,3-c]pyridine class of compounds
recently described by us,28 all other known agonists of TLR8,
such as the imidazoquinolines (for instance, 1),25,43 thiazolo-
quinolines (CL075, 2),27,44−47 and a 2-aminobenzazepine
derivative (VTX-2337, 3),48 display mixed TLR7/TLR8
agonism, with the sole exception of VTX-294,49 whose
complete structure has not been reported. The structures of
these mixed agonists are shown in Figure 1. The 2,3-diamino-
furo[2,3-c]pyridines are atypical and noncanonical in that
although they activate TLR8-dependent NF-κB signaling, they
are devoid of proinflammatory cytokine-inducing activities.28
a
Reagents: (i) I2, KI, NaOH; (ii) Pd(PPh3)4, CuI, alkyne, Et3N/
Figure 1. Representative TLR8/7 dual-agonistic heterocyclic small
CH3CN (1:3); (iii) m-CPBA, CHCl3; and (iv) (a) benzoyl isocyanate,
molecules.
CH2Cl2 and (b) NaOCH3, MeOH.
B
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Scheme 2. Syntheses of C2-alkylfuro[3,2-c]quinoline
a
analogues
a
Reagents: (i) I2, KI, NaOH; (ii) Pd(PPh3)4, CuI, alkyne, Et3N/
CH3CN (1:3); (iii) m-CPBA, CHCl3; and (iv) (a) benzoyl isocyanate,
CH2Cl2 and (b) NaOCH3, MeOH.
hydroxyquinoline proceeded uneventfully using reported
methods (Schemes 1 and 2, respectively).51 One-pot
Sonogashira coupling with a variety of alkynes followed by 5-
endo-dig cyclization yielded compounds the 2-substituted
furo[2,3-c]quinolines 6a−y (Scheme 1) and the regioisomeric
furo[3,2-c]quinolines 11a−c (Scheme 2) in good yields. Target
compounds 8a−y and 13a−c bearing 4-amino groups were
obtained using conventional procedures described previ-
ously.23,27,44 Compounds 8a−y and 13a−c were screened in
a panel of reporter-gene assays for human TLR2, -3, -4, -5, -7,
-8, and -9 and NOD1, -2 modulatory activities. We were
fortunate that a distinct activity profile was observed in the very
first set of compounds that were synthesized. Compounds 8b−f
showed pure TLR8-agonistic activity, with maximal potency
exhibited by 8d with a C2-butyl group (EC50 = 1.6 μM, Figure
2); shorter (8b, 8c) and longer (8e, 8f) homologues displayed
lower agonistic potencies, with shortest analogue 8a being
inactive (Figure 2). The dose−response profiles show
characteristic biphasic responses (dose-dependent activation
followed by the apparent suppression of NF-κB translocation,
Figure 2), as we had previously observed in several chemo-
types.23,28,52 We verified that the apparent suppression was not
due to cytotoxicity using LDH release and mitochondrial redox-
based assays, as described earlier by us.53,54
The SAR pattern with maximal activity conferred by a C2-
butyl group is virtually identical to that found in TLR7-active
imidazoquinolines23 and the TLR8/7-agonistic thiazoloquino-
lines,27 but unlike the thiazoloquinolines,27 8d was devoid of
TLR7-stimulatory activities in primary screens (Figure 2). The
exquisite selectivity for TLR8 was confirmed in secondary
screens using ex vivo whole human blood and PBMC models.17
We had earlier shown that proinflammatory cytokine induction
(TNF-α, IL-1β, IL-6, and IL-8) is a consequence of TLR8
activation.18 Importantly, Th1-biasing IL-12 and IL-18
induction is also TLR8-dependent, whereas IFN-α production
is TLR7-mediated.24,52 Therefore, we examined the cytokine-
and interferon-induction profiles of 8d, employing thiazoloqui-
noline 2 as a reference compound. Unlike the 2,3-diamino-
furo[2,3-c]pyridines,28 8d was observed to induce TNF-α, IL-
1β, IL-6, and IL-8 in a dose-dependent manner, albeit with a
lower potency than 2 (Figure 3). Compound 8d induced IL-12
and IL-18 but was bereft of IFN-α-inducing properties (Figure
4). The selectivity of 8d for TLR8 was also reflected in the
absence of natural killer lymphocyte activation17,18 in human
whole blood models, as assessed by cluster of differentiation 69
Figure 2. Dose−response profiles of human TLR8 (panel A) and
human TLR7 (panel B) agonism by select C2-alkyl furo[2,3-
c]quinolines. Error bars represent standard deviations obtained from
quadruplicates. Dual TLR7/8-agonistic compounds 1 and 2 were used
as comparators. Potency of TLR8 agonism (EC50 values) of a
homologous series of C2-alkyl analogues (panel C). Compound 8a
was inactive.
(CD69) expression (Figure 5), which we had previously shown
to be TLR7-dependent.17,18 Consistent with the above findings,
transcriptomal profiling experiments showed strong induction
of proinflammatory cytokine transcripts (including IL-12 and
IL-18) by both 2 and 8d, but IFN-α transcription was induced
by only 2 owing to its dual TLR8/7-agonistic activity (Table 1).
These data collectively confirm the selectivity of 8d for human
TLR8.
Given that 8d represents an optimized compound in a
uniquely TLR8-specific chemotype, we also profiled its
cytokine- and chemokine-inducing properties using a 41-
analyte multiplexed assay by comparing 8d to a variety of
TLR agonists. We found that of the 41 analytes analyzed both
8d and 2 induced granulocyte colony-stimulating factor (G-
CSF), growth-related oncogene (GRO), and macrophage
inflammatory proteins 1-α and -β (MIP-1α, MIP-1β), whereas
highly potent, pure TLR7-agonistic imidazoquinoline (1-
benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine) that we
identified in our earlier SAR studies23 did not (Figure 6),
indicating that this set of analytes could be useful in
distinguishing TLR8-specific responses.
Distinct hints that the SAR in the 2-substituted furo[2,3-
c]quinolines was much more stringent compared to the
thiazoloquinolines27 were evident from the outset. C2
C
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Figure 5. Absence of CD69 upregulation in human natural killer cells
by 8d. Flow cytometry data (inset) with gating on lymphocytes
showing CD3+CD56− (T cells, quadrant A), CD3−CD56− (nominal B
cells, quadrant B), CD3+CD56+ (cytokine-induced killer cells,
quadrant C), and CD3−CD56+ (natural killer cells, quadrant D).
the alkyl group (8w), but none of these compounds were active
in any of our primary screens.
Because we were mindful of our earlier observation that
regioisomerism in the imidazoquinolines resulted in a switch
from agonistic to antagonistic activities,22,55 we also synthesized
key regioisomeric furo[3,2-c]quinolines 13a−c (Scheme 2);
these compounds were quiescent, displaying neither stimula-
tory nor inhibitory activity in primary screens. Given the
fastidious structural requirements at the C2 position, we next
synthesized a furo[2,3-c]isoquinoline with a C2-butyl sub-
stituent (18, Scheme 3) to explore the chemical space beyond
the fused-quinoline chemotypes, but we were disappointed that
18 was also inactive.
Figure 3. Proinflammatory cytokine induction profiles of 8d in human
blood. 2 was used as reference/comparator compound. The mean of
duplicate values from a representative experiment is shown.
Reverting, therefore, to the C2-butyl furo[2,3-c]quinoline, we
interrogated the effect of introducing substituents at the C1
position. In particular, we desired to evaluate a C1-benzyl
substituent because this strategy had previously yielded highly
potent, TLR7-selective agonists in the imidazoquinoline
series.23 Electrophilic bromination of 7d furnished precursor
19 in good yield (Scheme 4), which was carried forward to
obtain both 1-bromo- and 1-benzyl-substituted analogues 21
and 24, respectively (Scheme 4). Both analogues were found to
be inactive.
As mentioned earlier, the 2,3-diamino-furo[2,3-c]pyridine
class of compounds recently described by us28 activate TLR8
but do not result in downstream cytokine production,
suggesting an apparent dissociation between receptor occu-
pancy and the signal-transduction events leading to cytokine
production, and it was of interest to compare the SAR that we
had gleaned from the furo[2,3-c]quinolines with select
analogues of the 4-amino, C2-alkyl furo[2,3-c]pyridine series.
Two such compounds (28a, 28b) were therefore synthesized
(Scheme 5). Both of these analogues retained TLR8-selective
agonistic activities but were substantially weaker (EC50 = 24.4
and 46.2 μM, respectively) than 8d (EC50 = 1.6 μM).
Although at first glance our attempts at exploring SAR in the
furo[2,3-c]quinolines had yielded a large number of inactive
compounds, we were delighted in no small measure that we
have finally arrived at pure TLR8 agonists that induce the
expected complement of TLR8-mediated, Th1-biasing cytokine
mediators but are devoid of TLR7-dependent IFN-α-inducing
properties. Just as we were concluding our studies on the
furo[2,3-c]quinolines, crystal structures of hTLR8 complexed
Figure 4. Dose−response profiles of IL-12p40 and IL-18 induction
and the absence of IFN-α by 8d. 2 was used as reference/comparator
compound. The mean of duplicate values from a representative
experiment is shown.
substituents with branched alkyl groups (8g−i) abrogated
activity, and analogues with cycloaliphatic (8j−8m) or aromatic
(8n, 8o) substituents were inactive, pointing to the intolerance
of steric bulk at the putative binding site(s). Because the crystal
structure of TLR8 was not available at the time, we forged on,
surmising that the introduction of polar H-bond donors (or
acceptors) on the C2 alkyl group may help us to understand
better the binding-site interactions. Therefore, we examined a
number of analogues bearing hydroxyl groups at various
positions along the C2-alkyl chain (8p−8v), compounds with
C2 substituents terminating with a primary amine (8x, 8y), and
an analogue with an ether (H-bond acceptor) incorporated in
D
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Table 1. Transcriptomal Profiling of 8d in Human PBMCs
Log(FC) Log(FC)
2 vs neg. 8d vs neg.
gene
probeset ID
208375_at
symbol
gene title
ctrl.
ctrl.
IFNA1
interferon, alpha 1
7.38297
5.36195
0.5418
208344_x_at IFNA1 /// interferon, alpha 1///13
IFNA13
0.85859
208261_x_at IFNA10
211405_x_at IFNA17
211145_x_at IFNA21
interferon, alpha 10
interferon, alpha 17
interferon, alpha 21
interferon, alpha 5
interferon, alpha 7
interferon, alpha 8
interferon, gamma
5.87577
4.25712
4.96822
7.03922
5.77059
4.18566
5.60123
0.88813
0.7771
0.81187
0.58271
1.22985
0.80266
0.61895
4.54852
1.99629
214569_at
IFNA5
208259_x_at IFNA7
207932_at
210354_at
204470_at
IFNA8
IFNG
CXCL1
chemokine (C-X-C
motif) ligand 1
209774_x_at CXCL2
chemokine (C-X-C
motif) ligand 2
2.83012
1.47026
1.51721
1.88404
7.689
2.8543
Figure 6. Disparate responses in select analytes (out of 41 analytes) in
human PBMCs. G-CSF, GRO, MIP-1α, and MIP-1β are induced by
8d (and 2, data not shown) but not by a pure TLR7-agonistic
imidazoquinoline. Dose responses represent the percent of the
maximal response (G-CSF, 622 pg/mL; GRO, 515 pg/mL; MIP-1α,
10 780 pg/mL; and MIP-1β, 10 374 pg/mL). The mean of duplicate
values from a representative experiment is shown. Analytes that were
below the detection limits for the negative controls (medium alone)
are not shown for clarity.
207850_at
215101_s_at
203915_at
204533_at
210163_at
211122_s_at
CXCL3
CXCL5
CXCL9
CXCL10
CXCL11
CXCL11
chemokine (C-X-C
motif) ligand 3
1.75638
2.39582
0.81365
6.42653
6.50285
6.56354
chemokine (C-X-C
motif) ligand 5
chemokine (C-X-C
motif) ligand 9
chemokine (C-X-C
motif) ligand 10
chemokine (C-X-C
motif) ligand 11
8.89003
9.00636
chemokine (C-X-C
motif) ligand 11
Scheme 3. Syntheses of C2-Alkylfuro[2,3-c]isoquinoline
a
Analogues
210118_s_at
205067_at
IL1A
interleukin 1, alpha
interleukin 1, beta
4.67389
2.45708
3.97769
4.82369
2.44858
3.66761
IL1B
216243_s_at
IL1RN
interleukin 1 receptor
antagonist
207538_at
205207_at
207901_at
IL4
interleukin 4
interleukin 6
2.20255
7.85102
5.84181
2.24232
7.67767
3.26706
IL6
IL12B
interleukin 12B (natural
killer cell stimulatory
factor 2)
207844_at
205992_s_at
209827_s_at
206295_at
207072_at
IL13
interleukin 13
interleukin 15
interleukin 16
interleukin 18
1.57695
1.16516
2.04086
1.06962
IL15
IL16
−1.7103 −0.64723
2.64035
IL18
2.40756
IL18RAP
interleukin 18 receptor
accessory protein
1.12674
0.51353
a
224071_at
222974_at
220054_at
IL20
interleukin 20
interleukin 22
0.31683
2.16422
4.73191
0.81046
0.38534
4.96526
Reagents: (i) I2, KI, NaOH; (ii) Pd(PPh3)4, CuI, alkyne, Et3N/
IL22
CH3CN (1:3); (iii) m-CPBA, CHCl3; and (iv) (a) benzoyl isocyanate,
CH2Cl2 and (b) NaOCH3, MeOH.
IL23A
interleukin 23, alpha
subunit p19
206569_at
205926_at
IL24
interleukin 24
−0.6150 −0.00233
IL27RA
interleukin 27 receptor,
alpha
0.20872
1.65564
1.13284
0.16269
0.59624
0.78929
used for docking studies. We employed induced-fit meth-
ods57,58 in which the receptor sites were allowed conforma-
tional and torsional flexibility to simulate realistic ligand−
receptor interactions to account for conformational changes in
the binding site residues. Compounds 2 and 8d occupy the
same binding pocket formed by both of the TLR8 protomers
(Figure 7A,B), with the binding geometry of these compounds
and interacting residues being virtually identical (Figure 7C,D).
Strong ionic H bonds (salt bridges) are observed between the
C4 amine of both 2 and 8d with Asp543 of protomer B, with
additional stabilization derived from an H bond between
Thr574 (protomer B) and either the N3 atom of the thiazole
ring of 2 or the oxygen atom of the furanyl ring of 8d. π−π
interactions of the quinoline moiety of 2 and 8d (Phe405/
Tyr353) as well as hydrophobic interactions of the C2-alkyl
group (Phe346/Ile403/Gly376) occur exclusively with residues
in protomer A (Figure 7C,D). All of these interactions appear
indispensable. The butyl group of 8d allows for excellent
1552915_at
244261_at
IL28A
interleukin 28A
(interferon, lambda 2)
IL28RA
interleukin 28 receptor,
alpha (interferon,
lambda receptor)
1552917_at
IL29
interleukin 29
(interferon, lambda 1)
1.02924
0.76252
203828_s_at
207113_s_at
206025_s_at
IL32
interleukin 32
0.35684
3.81754
4.28297
0.70521
3.3126
TNF
tumor necrosis factor
TNFAIP6
tumor necrosis factor,
alpha-induced
protein 6
4.17629
with mixed TLR7/TLR8-agonistic imidazoquinolines and
thiazoloquinolines were reported,56 allowing us to rationalize
our experimentally determined SAR on the pure TLR8-
agonistic furo[2,3-c]quinolines. The crystal structure of
human TLR8 in complex with 2 (PDB ID: 3W3K)56 was
E
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a
Scheme 4. Syntheses of C1-Substituted Furo[2,3-c]quinoline Analogues
a
Reagents: (i) Br2, CH2Cl2; (ii) m-CPBA CHCl3; (iii) (a) benzoyl isocyanate, CH2Cl2, (b) NaOCH3, MeOH; and (iv) Pd(dppf)Cl2, 2-benzyl-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Cs2CO3, 1,4-dioxane.
Scheme 5. Syntheses of C2-Alkylfuro[2,3-c]pyridine
a
Analogues
a
Reagents: (i) Pd(PPh3)4, CuI, alkyne, Et3N/CH3CN (1:3); (ii) m-
CPBA, CHCl3; and (iii) (a) benzoyl isocyanate, CH2Cl2 and (b)
NaOCH3, MeOH.
nonpolar and van der Waals contacts in the rather shallow
hydrophobic cavity, and homologues with increasing C2-chain
length (8e−8f) dock poorly because of kinked and sterically
unfavorable conformations of the alkyl group. Analogues with
cycloaliphatic (8j−8m) and aromatic substituents (8n, 8o) do
not fit in the hydrophobic pocket at all, leading to unrealistic
docked conformations (not shown). The cavity is lined entirely
with the side chains of hydrophobic residues, explaining why
even length-optimized analogues bearing polar groups at the
Figure 7. Induced-fit docking of ligands in human TLR8 (PDB ID:
3W3K). Both 2 and 8d occupy the same binding pocket (panel A).
Binding modes of the TLR7/8-dual active imidazoquinoline CL097
and 8d are superimposable (panel B). Binding-site interactions of 2,
C2 position, such as 8r, 8u, and 8v, do not display TLR8
8d, 13b, and 18, respectively (panels C−F).
agonism. The feeble activity of furopyridine compounds 28a
and 28b imply significant contributions to the binding free
energies by the π−π interactions of the quinoline moiety with
Tyr353 and Phe405. The imposition of additional steric bulk at
C1 (compound 24) is not tolerated in the cleft bounded by
Phe261 and Ser352 (Figure 7D). Not only is the pivotal ionic
H bond between the C4 amine of regioisomeric furo[3,2-
c]quinoline 13b and Asp543 weakened (3.8 Å) but also the
additional H bond between the oxygen atom of the furan ring
and Thr574 is lost, forcing 13b to bind to TLR8 in an inverted
fashion, with the C2-alkyl group facing the entrance to the
binding site (Figure 7E). Highly unfavorable H bonds are also
seen in isoquinoline analogue 18, especially with the loss of H
bonding of the furanyl oxygen (Figure 7F).
An exploration of furo[2,3-c]quinolines have yielded pure
TLR8 agonists that are expected to possess strong Th1-biasing
adjuvantic properties, as evidenced by prominent IL-12 and IL-
18 induction profiles, and are without IFN-α-inducing
properties, confirming their exquisite selectivity for human
TLR8. The recently published atomic structure of human
TLR8 has permitted a careful evaluation of SAR in this hitherto
uncharacterized chemotype and provides clear directions
F
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Journal of Medicinal Chemistry
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27.5, 22.5, 14.1. MS (ESI) calcd for C16H17NO (m/z), 239.13; found,
toward rational, structure-based ligand design. Immunization
studies employing neonatal animal models are being planned
with a view toward comparing the adjuvanticity of agonists with
pure TLR7- and TLR8-agonistic properties vis-a-vis com-
̀
pounds with dual TLR7/8-stimulatory activities.
240.14 [M + H]+.
1
2-Hexylfuro[2,3-c]quinoline (6f). White solid (70 mg, 75%). H
NMR (500 MHz, CDCl3) δ 9.08 (s, 1H), 8.20 (t, J = 8.3 Hz, 1H), 8.07
(dd, J = 8.1, 1.1 Hz, 1H), 7.66 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.60
(ddd, 1H), 6.90 (d, J = 0.6 Hz, 1H), 2.91 (t, J = 7.6 Hz, 2H), 1.87−
1.79 (m, 2H), 1.50−1.40 (m, 2H), 1.39−1.28 (m, 4H), 0.90 (t, J = 8.2,
5.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 163.2, 148.8, 144.3,
136.3, 131.4, 130.1, 127.3, 126.5, 123.7, 123.2, 101.0, 31.7, 29.0, 28.9,
27.8, 22.7, 14.2. MS (ESI) calcd for C17H19NO (m/z), 253.15; found,
254.16 [M + H]+.
EXPERIMENTAL SECTION
■
Chemistry. All of the solvents and reagents used were obtained
commercially and used as such unless noted otherwise. Moisture- or
air-sensitive reactions were conducted under a nitrogen atmosphere in
an oven-dried (120 °C) glass apparatus. The solvents were removed
under reduced pressure using standard rotary evaporators. Flash
column chromatography was carried out using RediSep Rf Gold high-
performance silica columns on a CombiFlash Rf instrument unless
otherwise mentioned, whereas thin-layer chromatography was carried
out on silica gel (200 μm) CCM precoated aluminum sheets. The
purity for all final compounds was confirmed to be greater than 97%
by LC−MS using a Zorbax Eclipse Plus 4.6 mm × 150 mm, 5 μm
analytical reverse-phase C18 column with H2O−isopropanol or H2O−
CH3CN gradients and an Agilent ESI-QTOF mass spectrometer
(mass accuracy of 3 ppm) operating in the positive ion (or negative
ion, as appropriate) acquisition mode.
2-Isobutylfuro[2,3-c]quinoline (6g). Liquid (120 mg, 72%). 1H
NMR (500 MHz, CDCl3) δ 9.12 (s, 1H), 8.25 (d, J = 8.2 Hz, 1H),
8.08 (d, J = 7.9 Hz, 1H), 7.69−7.64 (m, 1H), 7.63−7.58 (m, 1H), 6.91
(s, 1H), 2.79 (dd, J = 7.1, 0.5 Hz, 2H), 2.20 (dp, J = 13.6, 6.8 Hz, 1H),
1.03 (d, J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 162.4, 144.3,
136.4, 131.5, 130.2, 127.4, 126.6, 123.7, 102.0, 38.0, 28.1, 22.6. MS
(ESI) calcd for C15H15NO (m/z), 225.12; found, 226.13 [M + H]+.
2-(tert-Butyl)furo[2,3-c]quinoline (6h). White solid (69 mg, 83%).
1H NMR (500 MHz, CDCl3) δ 9.11 (s, 1H), 8.20 (d, J = 8.3 Hz, 1H),
8.08 (d, J = 7.9 Hz, 1H), 7.69−7.64 (m, 1H), 7.59 (t, J = 7.4 Hz, 1H),
6.89 (s, 1H), 1.47 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 170.7,
148.8, 144.3, 136.4, 131.2, 130.1, 127.3, 126.5, 123.6, 123.4, 98.2, 33.6,
29.1. MS (ESI) calcd for C15H15NO (m/z), 225.12; found, 226.14 [M
+ H]+.
Synthesis of 4-Iodoquinolin-3-ol (5). In an oven-dried round-
bottomed flask equipped with a stirring bar was placed 3-hydroxy
quinoline (1.0 g, 6.89 mmol) in 2N NaOH (20 mL). To this mixture,
a solution of iodine (8.27 mmol) in 20% aqueous potassium iodide
(20 mL) was added dropwise, and the mixture was stirred for 3 h at
room temperature. The mixture was then acidified with acetic acid, and
the precipitate was filtered and washed with water. After drying under
vacuum, 1.50 g of 5 was obtained, which was used without purification.
1H NMR (500 MHz, DMSO) δ 11.19 (s, 1H), 8.50 (s, 1H), 7.98−7.85
(m, 2H), 7.65−7.60 (m, 1H), 7.60−7.55 (m, 1H). 13C NMR (126
MHz, DMSO) δ 152.2, 142.6, 141.3, 130.9, 129.9, 129.2, 128.5, 126.6,
94.5. MS (ESI) calcd for C9H6INO (m/z), 270.95; found, 271.96 [M
+ H]+.
2-Isopentylfuro[2,3-c]quinoline (6i). Solid (65 mg, 74%). 1H NMR
(500 MHz, CDCl3) δ 9.10 (s, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.07 (d, J
= 7.9 Hz, 1H), 7.70−7.63 (m, 1H), 7.59 (t, 1H), 6.90 (s, 1H), 2.91 (t,
2H), 1.79−1.64 (m, 3H), 0.99 (d, J = 6.4 Hz, 6H). 13C NMR (126
MHz, CDCl3) δ 163.4, 148.8, 144.3, 136.3, 131.4, 130.2, 127.3, 126.5,
123.7, 123.2, 100.9, 36.7, 27.8, 26.8, 22.5. MS (ESI) calcd for
C16H17NO (m/z), 239.13; found, 240.14 [M + H]+.
2-Cyclopropylfuro[2,3-c]quinoline (6j). Solid (60 mg, 78%). 1H
NMR (500 MHz, CDCl3) δ 9.02 (d, J = 0.7 Hz, 1H), 8.17 (dd, J = 8.4,
0.6 Hz, 1H), 8.04 (ddd, J = 8.1, 1.5, 0.6 Hz, 1H), 7.66 (ddd, J = 8.4,
6.9, 1.5 Hz, 1H), 7.58 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.89−6.85 (m,
1H), 2.21−2.14 (m, 1H), 1.12 (dqd, J = 6.0, 2.4, 1.2 Hz, 4H). 13C
NMR (126 MHz, CDCl3) δ 164.0, 148.3, 144.4, 136.0, 131.7, 130.1,
127.4, 126.4, 123.7, 123.0, 99.4, 10.1, 8.5. MS (ESI) calcd for
C14H11NO (m/z), 209.10; found, 210.10 [M + H]+.
2-Cyclopentylfuro[2,3-c]quinoline (6k). White solid (72 mg, 82%).
1H NMR (500 MHz, CDCl3) δ 9.08 (s, 1H), 8.20 (d, J = 8.3 Hz, 1H),
8.07 (d, J = 7.7 Hz, 1H), 7.70−7.64 (m, 1H), 7.59 (t, J = 11.0, 3.8 Hz,
1H), 6.91 (s, 1H), 3.41−3.31 (m, 1H), 2.23−2.13 (m, 2H), 1.95−1.81
(m, 4H), 1.81−1.69 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 166.8,
144.3, 136.4, 131.3, 130.1, 127.3, 126.5, 123.7, 123.2, 99.6, 39.4, 32.1,
25.6. MS (ESI) calcd for C16H15NO (m/z), 237.11; found, 238.13 [M
+ H]+.
General Procedure for the Sonogashira Reaction. To a stirred
solution of 4-iodoquinolin-3-ol in acetonitrile/triethylamine (2:1)
were added the appropriate alkyne (0.553 mmol), Pd(PPh3)4 (0.018
mmol), and CuI (0.018 mmol). The resulting reaction mixture was
stirred at 70 °C under a nitrogen atmosphere for 12 h. After the
completion of the reaction (monitored by TLC), the reaction mixture
was diluted with water and extracted with ethylacetate (3 × 10 mL).
The combined organic layer was dried over Na2SO4 and concentrated
under reduced pressure, and the crude material was purified by flash
chromatography using CH2Cl2/MeOH as the eluent.
1
2-Propylfuro[2,3-c]quinoline (6c). Yellow solid (60 mg, 78%). H
NMR (500 MHz, CDCl3) δ 9.08 (s, 1H), 8.20 (d, J = 8.2 Hz, 1H),
8.07 (dd, J = 8.1, 1.2 Hz, 1H), 7.67 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.60
(ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.91 (d, J = 0.8 Hz, 1H), 2.91−2.86 (m,
2H), 1.91−1.82 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 163.0, 148.8, 144.3, 136.3, 131.4, 130.1, 127.3, 126.5,
123.7, 123.2, 101.1, 30.8, 21.2, 13.9. MS (ESI) calcd for C14H13NO
(m/z), 211.10; found, 212.11 [M + H]+.
2-(Cyclopentylmethyl)furo[2,3-c]quinoline (6l). Solid (71 mg,
1
757%). H NMR (500 MHz, CDCl3) δ 9.10 (s, 1H), 8.22 (d, J =
8.2 Hz, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.67 (t, J = 7.0 Hz, 1H), 7.60 (t,
1H), 6.91 (s, 1H), 2.91 (d, J = 7.4 Hz, 2H), 2.38 (dt, J = 15.4, 7.8 Hz,
1H), 1.87 (m, 2H), 1.74−1.65 (m, 2H), 1.64−1.57 (m, 2H), 1.32 (m,
2H). 13C NMR (126 MHz, CDCl3) δ 163.0, 144.3, 136.4, 131.5, 130.2,
127.4, 126.5, 123.7, 101.5, 38.9, 34.9, 32.7, 25.2. MS (ESI) calcd for
C17H17NO (m/z), 251.13; found, 252.14 [M + H]+.
1
2-Butylfuro[2,3-c]quinoline (6d). Yellow solid (70 mg, 83%). H
NMR (500 MHz, CDCl3) δ 9.08 (d, J = 0.5 Hz, 1H), 8.19 (dd, J = 8.4,
0.5 Hz, 1H), 8.09−8.05 (m, 1H), 7.66 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H),
7.60 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.90 (d, J = 0.8 Hz, 1H), 2.92 (t,
2H), 1.82 (ddd, J = 15.2, 8.5, 6.7 Hz, 2H), 1.51−1.42 (m, 2H), 0.99 (t,
J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 163.2, 148.8, 144.3,
136.4, 131.4, 130.1, 127.3, 126.5, 123.7, 123.2, 101.0, 29.9, 28.5, 22.4,
13.9. MS (ESI) calcd for C15H15NO (m/z), 225.11; found, 226.13 [M
+ H]+.
2-(Cyclohexylmethyl)furo[2,3-c]quinoline (6m). Solid (170 mg,
1
87%). H NMR (500 MHz, CDCl3) δ 9.10 (s, 1H), 8.21 (d, J = 8.3
Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.72−7.63 (m, 1H), 7.59 (t, J = 7.4
Hz, 1H), 6.89 (s, 1H), 2.78 (d, J = 6.9 Hz, 2H), 1.89−1.64 (m, 6H),
1.33−1.12 (m, 3H), 1.05 (qd, J = 12.4, 3.1 Hz, 2H). 13C NMR (126
MHz, CDCl3) δ 162.1, 148.8, 144.23, 136.4, 131.4, 130.1, 127.3, 126.5,
123.7, 123.2, 102.0, 37.3, 36.7, 33.3, 26.4, 26.2. MS (ESI) calcd for
C18H19NO (m/z), 265.15; found, 266.16 [M + H]+.
1
2-Pentylfuro[2,3-c]quinoline (6e). White solid (70 mg, 79%). H
1
NMR (500 MHz, CDCl3) δ 9.08 (d, J = 0.7 Hz, 1H), 8.20 (dd, J = 8.4,
0.6 Hz, 1H), 8.07 (ddd, J = 8.1, 1.5, 0.6 Hz, 1H), 7.66 (ddd, J = 8.4,
6.9, 1.5 Hz, 1H), 7.60 (ddd, J = 8.1, 6.9, 1.3 Hz, 1H), 6.91−6.90 (m,
1H), 2.91 (t, J = 7.2 Hz, 2H), 1.88−1.80 (m, 2H), 1.46−1.35 (m, 4H),
0.93 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 163.3, 148.8,
144.3, 136.4, 131.4, 130.1, 127.4, 126.5, 123.7, 123.2, 101.0, 31.5, 28.8,
2-Phenylfuro[2,3-c]quinoline (6n). White solid (65 mg, 72%). H
NMR (500 MHz, CDCl3) δ 9.20 (d, 1H), 8.23 (dd, J = 8.3, 0.7 Hz,
1H), 8.17 (ddd, J = 8.0, 1.5, 0.5 Hz, 1H), 8.01−7.97 (m, 2H), 7.71
(ddd, J = 8.4, 6.9, 1.6 Hz, 1H), 7.65 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H),
7.55−7.50 (m, 3H), 7.47−7.43 (m, 1H). 13C NMR (126 MHz,
CDCl3) δ 158.8, 148.9, 144.5, 136.7, 131.6, 130.3, 129.8, 129.8, 129.2,
G
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127.7, 126.9, 125.6, 123.7, 123.2, 99.9. MS (ESI) calcd for C17H11NO
mL). The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure, and the crude material was
purified by flash chromatography.
(m/z), 245.10; found, 246.10 [M + H]+.
1
Furo[2,3-c]quinolin-2-ylmethanol (6p). Solid (56 mg, 77%). H
2-Methylfuro[2,3-c]quinoline 5-oxide (7b). White solid (80 mg,
74%). H NMR (500 MHz, CDCl3) δ 8.86 (s, 1H), 8.84 (dd, 1H),
NMR (500 MHz, MeOD) δ 9.07 (s, 1H), 8.26 (dd, J = 8.0, 1.2 Hz,
1H), 8.12 (d, J = 8.2 Hz, 1H), 7.77−7.71 (m, 1H), 7.71−7.66 (m,
1H), 7.39 (s, 1H), 4.84 (s, 2H). 13C NMR (126 MHz, MeOD) δ
163.5, 150.2, 144.8, 137.2, 133.0, 129.7, 129.1, 128.3, 125.0, 124.6,
103.3, 58.1. MS (ESI) calcd for C12H9NO2 (m/z), 199.10; found,
200.07 [M + H]+.
1
8.08−8.03 (m, 1H), 7.76−7.68 (m, 2H), 6.87 (t, 1H), 2.59 (d, J = 1.0
Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 159.7, 147.2, 138.8, 129.0,
128.6, 124.5, 124.3, 122.7, 121.0, 102.2, 14.5. MS (ESI) calcd for
C12H9NO2 (m/z), 199.10; found, 200.06 [M + H]+.
2-Propylfuro[2,3-c]quinoline 5-oxide (7c). White solid (78 mg,
72%). 1H NMR (500 MHz, CDCl3) δ 8.88−8.82 (m, 2H), 8.08−8.04
(m, 1H), 7.75−7.68 (m, 2H), 6.86 (dd, J = 1.7, 0.8 Hz, 1H), 2.88−
2.83 (m, 2H), 1.88−1.79 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR
(126 MHz, CDCl3) δ 163.7, 147.1, 138.8, 128.9, 128.5, 124.4, 124.2,
124.1, 122.8, 121.1, 101.4, 30.7, 21.1, 13.9. MS (ESI) calcd for
C14H13NO2 (m/z), 227.10; found, 228.10 [M + H]+.
2-Butylfuro[2,3-c]quinoline 5-Oxide (7d). White solid (75 mg,
70%). 1H NMR (500 MHz, CDCl3) δ 8.86 (d, J = 0.5 Hz, 1H), 8.85−
8.83 (m, 1H), 8.08−8.04 (m, 1H), 7.75−7.68 (m, 2H), 6.86 (d, J = 0.9
Hz, 1H), 2.90−2.86 (m, 2H), 1.82−1.76 (m, 2H), 1.46 (dq, J = 14.7,
7.4 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
164.0, 147.1, 138.7, 128.9, 128.5, 124.5, 124.2, 124.2, 122.8, 121.0,
101.3, 29.8, 28.5, 22.4, 13.9. MS (ESI) calcd for C15H15NO2 (m/z),
241.11; found, 242.12 [M + H]+.
2-Pentylfuro[2,3-c]quinoline 5-Oxide (7e). White solid (77 mg,
72%). 1H NMR (500 MHz, CDCl3) δ 8.87−8.86 (m, 1H), 8.86−8.83
(m, 1H), 8.09−8.05 (m, 1H), 7.76−7.68 (m, 2H), 6.86 (d, J = 0.8 Hz,
1H), 2.87 (t, J = 7.4 Hz, 2H), 1.84−1.78 (m, 2H), 1.40 (qd, J = 4.6, 1.7
Hz, 2H), 0.93 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
164.0, 147.1, 138.7, 128.9, 128.5, 124.5, 124.2, 124.2, 122.8, 121.1,
101.3, 31.5, 28.8, 27.4, 22.5, 14.1. MS (ESI) calcd for C16H17NO2 (m/
z), 255.12; found, 256.14 [M + H]+.
2-Hexylfuro[2,3-c]quinoline 5-Oxide (7f). White solid (105 mg
82%). 1H NMR (500 MHz, CDCl3) δ 8.86 (d, J = 0.5 Hz, 1H), 8.86−
8.82 (m, 1H), 8.08−8.05 (m, 1H), 7.75−7.68 (m, 2H), 6.86 (d, J = 0.9
Hz, 1H), 2.87 (t, J = 7.3 Hz, 2H), 1.80 (dt, J = 15.2, 7.5 Hz, 2H),
1.47−1.39 (m, 2H), 1.36−1.30 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 164.0, 147.1, 138.8, 128.9, 128.5, 124.5,
124.2, 124.2, 122.8, 121.1, 101.3, 31.6, 29.0, 28.8, 27.7, 22.7, 14.2. MS
(ESI) calcd for C17H19NO2 (m/z), 269.14; found, 270.15 [M + H]+.
2-Isobutylfuro[2,3-c]quinoline 5-Oxide (7g). Solid (100 mg, 78%).
1H NMR (500 MHz, CDCl3) δ 8.87 (s, 1H), 8.85 (dt, J = 7.2, 3.9 Hz,
1H), 8.09−8.06 (m, 1H), 7.76−7.69 (m, 2H), 6.88 (d, J = 0.7 Hz,
1H), 2.76 (dd, J = 7.1, 0.5 Hz, 2H), 2.22−2.12 (m, 1H), 1.03 (d, J =
6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 163.0, 147.2, 138.8,
128.9, 128.5, 124.5, 124.3, 124.1, 122.8, 121.1, 102.3, 37.9, 28.0, 22.6.
MS (ESI) calcd for C15H15NO2 (m/z), 241.11; found, 242.13 [M +
H]+.
1
2-(Furo[2,3-c]quinolin-2-yl)ethanol (6q). Solid (58 mg, 74%). H
NMR (500 MHz, MeOD) δ 9.02 (s, 1H), 8.23 (dd, 1H), 8.10 (dd, J =
8.3, 0.5 Hz, 1H), 7.71 (ddd, J = 8.5, 7.0, 1.6 Hz, 1H), 7.67 (ddd, J =
8.1, 7.0, 1.3 Hz, 1H), 7.26 (d, J = 0.7 Hz, 1H), 4.01 (t, J = 6.4 Hz, 2H),
3.17 (td, J = 6.4, 0.6 Hz, 2H). 13C NMR (126 MHz, MeOD) δ 162.9,
149.9, 144.7, 136.7, 133.6, 129.6, 128.9, 128.0, 125.1, 124.4, 103.5,
60.6, 33.1. MS (ESI) calcd for C13H11NO2 (m/z), 213.08; found,
214.09 [M + H]+.
4-(Furo[2,3-c]quinolin-2-yl)butan-1-ol (6s). Solid (68 mg, 77%).
1H NMR (500 MHz, MeOD) δ 8.99 (s, 1H), 8.22−8.18 (m, 1H), 8.09
(dd, J = 8.4, 0.6 Hz, 1H), 7.70 (ddd, J = 8.4, 6.9, 1.6 Hz, 1H), 7.65
(ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.17 (d, J = 0.8 Hz, 1H), 3.63 (t, J = 6.4
Hz, 2H), 2.98 (t, 2H), 1.96−1.88 (m, 2H), 1.71−1.63 (m, 2H). 13C
NMR (126 MHz, MeOD) δ 165.5, 149.8, 144.7, 136.6, 133.6, 129.5,
128.9, 128.0, 125.1, 124.3, 102.4, 62.4, 33.0, 29.2, 25.2. MS (ESI) calcd
for C15H15NO2 (m/z), 241.11; found, 242.12 [M + H]+.
2-(Furo[2,3-c]quinolin-2-yl)propan-2-ol (6t). White solid (60 mg,
72%). 1H NMR (500 MHz, MeOD) δ 9.07 (d, J = 0.6 Hz, 1H), 8.29−
8.24 (m, 1H), 8.14−8.10 (m, 1H), 7.73 (ddd, J = 8.5, 7.0, 1.6 Hz, 1H),
7.68 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.36 (d, J = 0.8 Hz, 1H), 1.72 (s,
6H). 13C NMR (126 MHz, MeOD) δ 169.7, 149.9, 144.8, 137.1,
133.0, 129.7, 129.0, 128.2, 125.0, 124.6, 100.6, 70.1, 29.1. MS (ESI)
calcd for C14H13NO2 (m/z), 227.10; found, 228.11 [M + H]+.
1-(Furo[2,3-c]quinolin-2-yl)propan-2-ol (6v). White solid (70 mg,
84%). 1H NMR (500 MHz, MeOD) δ 9.04 (d, J = 0.5 Hz, 1H), 8.27−
8.23 (m, 1H), 8.11 (dd, J = 8.4, 0.6 Hz, 1H), 7.73 (ddd, J = 8.5, 7.0, 1.6
Hz, 1H), 7.68 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.28 (d, J = 0.7 Hz, 1H),
4.32−4.25 (m, 1H), 3.09 (d, J = 6.2 Hz, 2H), 1.31 (d, J = 6.2 Hz, 3H).
13C NMR (126 MHz, MeOD) δ 163.0, 149.9, 144.6, 136.7, 133.7,
129.5, 129.0, 128.1, 125.1, 124.4, 104.0, 67.1, 39.2, 23.4. MS (ESI)
calcd for C14H13NO2 (m/z), 227.10; found, 228.11 [M + H]+.
2-(2-Ethoxyethyl)furo[2,3-c]quinoline (6w). Solid (70 mg, 77%).
1H NMR (500 MHz, CDCl3) δ 9.10 (s, 1H), 8.21 (d, J = 8.3 Hz, 1H),
8.08 (dd, J = 8.1, 1.1 Hz, 1H), 7.68 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H),
7.63−7.59 (m, 1H), 7.02 (d, 1H), 3.87 (t, J = 6.6 Hz, 2H), 3.57 (q, J =
7.0 Hz, 2H), 3.20 (td, J = 6.6, 0.8 Hz, 2H), 1.23 (t, J = 7.0 Hz, 3H).
13C NMR (126 MHz, CDCl3) δ 160.1, 148.8, 144.3, 136.3, 131.4,
130.1, 127.3, 126.6, 123.7, 123.2, 102.3, 68.0, 66.7, 29.8, 15.3. MS
(ESI) calcd for C15H15NO2 (m/z), 241.11; found, 242.11 [M + H]+.
2-(Furo[2,3-c]quinolin-2-ylmethyl)isoindoline-1,3-dione (6x).
2-(tert-Butyl)furo[2,3-c]quinoline 5-Oxide (7h). White solid (72
1
mg, 68%). H NMR (500 MHz, CDCl3) δ 8.88 (d, J = 0.6 Hz, 1H),
1
White solid (33 mg, 61%). H NMR (500 MHz, DMSO) δ 9.20 (d,
8.85 (dd, J = 7.8, 2.1 Hz, 1H), 8.09−8.06 (m, 1H), 7.75−7.69 (m,
2H), 6.85 (d, J = 0.9 Hz, 1H), 1.45 (s, 9H). 13C NMR (126 MHz,
CDCl3) δ 171.5, 147.1, 138.7, 128.9, 128.5, 124.6, 124.2, 124.1, 123.0,
121.1, 98.6, 29.0. MS (ESI) calcd for C15H15NO2 (m/z), 241.11;
found, 242.12 [M + H]+.
2-Isopentylfuro[2,3-c]quinoline 5-Oxide (7i). Solid (60 mg, 77%).
1H NMR (500 MHz, MeOD) δ 9.13 (d, J = 0.5 Hz, 1H), 8.72 (dd, J =
8.1, 1.6 Hz, 1H), 8.37−8.34 (m, 1H), 7.90−7.83 (m, 2H), 7.28 (d, J =
0.8 Hz, 1H), 3.01−2.96 (m, 2H), 1.78−1.72 (m, 2H), 1.72−1.65 (m,
1H), 1.01 (d, J = 6.5 Hz, 6H). 13C NMR (126 MHz, MeOD) δ 167.7,
147.9, 138.8, 130.6, 130.5, 128.7, 126.7, 126.0, 124.0, 120.8, 102.8,
37.6, 28.9, 27.5, 22.7. MS (ESI) calcd for C16H17NO2 (m/z), 255.12;
found, 256.13 [M + H]+.
2-Cyclopropylfuro[2,3-c]quinoline 5-Oxide (7j). Solid (40 mg
74%). 1H NMR (500 MHz, MeOD) δ 9.11−8.99 (m, 1H), 8.73 (t, J =
13.1 Hz, 1H), 8.36−8.30 (m, 1H), 7.96−7.78 (m, 2H), 7.24 (s, 1H),
2.35−2.24 (m, 1H), 1.27−1.16 (m, 2H), 1.15−1.09 (m, 2H). 13C
NMR (126 MHz, MeOD) δ 168.84, 130.7, 130.3, 129.8, 129.3, 126.0,
125.1, 123.6, 120.7, 100.9, 10.6, 9.2. MS (ESI) calcd for C14H11NO2
(m/z), 225.10; found, 226.09 [M + H]+.
J = 0.5 Hz, 1H), 8.32 (d, 1H), 8.11 (dd, J = 8.3, 0.7 Hz, 1H), 7.96 (dd,
J = 5.5, 3.0 Hz, 2H), 7.89 (dd, J = 5.5, 3.1 Hz, 2H), 7.75−7.64 (m,
3H), 5.11 (d, J = 0.4 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 167.3,
155.6, 148.3, 143.7, 136.6, 134.8, 131.6, 130.1, 129.6, 127.7, 126.9,
124.1, 123.5, 122.6, 103.8, 34.9. MS (ESI) calcd for C20H12N2O3 (m/
z), 328.08; found, 329.10 [M + H]+.
2-(2-(Furo[2,3-c]quinolin-2-yl)ethyl)isoindoline-1,3-dione (6y).
1
White solid (65 mg, 77%). H NMR (500 MHz, DMSO) δ 9.13 (s,
1H), 8.23 (dd, J = 8.0, 1.4 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.90−
7.79 (m, 4H), 7.73−7.68 (m, 1H), 7.68−7.63 (m, 1H), 7.44 (s, 1H),
4.03 (t, J = 6.9 Hz, 2H), 3.30 (t, J = 6.9 Hz, 2H). 13C NMR (126 MHz,
DMSO) δ 167.7, 158.9, 148.5, 143.7, 136.2, 134.5, 131.6, 130.4, 129.5,
127.5, 126.7, 124.0, 123.2, 122.6, 103.1, 36.0, 27.3. MS (ESI) calcd for
C21H14N2O3 (m/z), 342.10; found, 343.12 [M + H]+.
General Procedure for N-Oxidation. To a stirred solution of
substrate (0.53 mmol) in CHCl3 was added m-CPBA (1.06 mmol).
The resulting reaction mixture was stirred at room temperature for 4 h.
After the completion of the reaction (monitored by TLC), the reaction
mixture was diluted with water and extracted with CH2Cl2 (3 × 10
H
dx.doi.org/10.1021/jm400694d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2-Cyclopentylfuro[2,3-c]quinoline 5-Oxide (7k). White solid (77
mg, 72%). H NMR (500 MHz, MeOD) δ 9.12 (d, J = 0.4 Hz, 1H),
MS (ESI) calcd for C15H15NO3 (m/z), 257.10; found, 258.11 [M +
H]+.
1
2-(2-Hydroxypropan-2-yl)furo[2,3-c]quinoline 5-Oxide (7t). White
solid (63 mg 74%). 1H NMR (500 MHz, MeOD) δ 9.17 (s, 1H), 8.73
(d, 1H), 8.38 (dd, 1H), 7.92−7.83 (m, 2H), 7.42 (d, 1H), 1.70 (s,
6H). 13C NMR (126 MHz, MeOD) δ 171.2, 148.0, 139.0, 130.8,
130.7, 127.9, 127.0, 126.0, 124.3, 120.9, 101.3, 70.0, 29.0. MS (ESI)
calcd for C14H13NO3 (m/z), 243.10; found, 244.10 [M + H]+.
2-(1-Hydroxy-3-methylbutyl)furo[2,3-c]quinoline 5-Oxide (7u).
8.71 (dd, 1H), 8.35 (dd, 1H), 7.89−7.81 (m, 2H), 7.29 (d, J = 0.7 Hz,
1H), 3.47−3.39 (m, 1H), 2.26−2.14 (m, 2H), 1.96−1.85 (m, 4H),
1.81−1.72 (m, 2H). 13C NMR (126 MHz, MeOD) δ 171.0, 147.9,
138.8, 130.6, 130.5, 128.7, 126.7, 126.0, 124.0, 120.8, 101.5, 40.5, 32.8,
26.4. MS (ESI) calcd for C16H15NO2 (m/z), 253.11; found, 254.12 [M
+ H]+.
2-(Cyclopentylmethyl)furo[2,3-c]quinoline 5-Oxide (7l). White
1
1
Solid (60 mg, 70%). H NMR (500 MHz, MeOD) δ 9.18 (s, 1H),
solid (75 mg, 70%). H NMR (500 MHz, MeOD) δ 9.13 (s, 1H),
8.74 (dd, 1H), 8.40 (dd, 1H), 7.92−7.85 (m, 2H), 7.46 (s, 1H), 4.98
(dd, J = 8.5, 5.1 Hz, 1H), 1.91−1.85 (m, 2H), 1.84−1.78 (m, 1H),
1.02 (dd, J = 6.3, 3.0 Hz, 6H). 13C NMR (126 MHz, MeOD) δ 168.3,
148.1, 139.0, 130.7, 130.7, 127.9, 127.0, 126.0, 124.3, 120.9, 102.8,
66.9, 45.7, 25.7, 23.6, 22.3. MS (ESI) calcd for C16H17NO3 (m/z),
271.12; found, 272.12 [M + H]+.
8.74−8.70 (m, 1H), 8.36 (dd, J = 7.5, 1.8 Hz, 1H), 7.91−7.82 (m,
2H), 7.29 (d, J = 0.5 Hz, 1H), 2.96 (d, J = 7.4 Hz, 2H), 2.44−2.37 (m,
1H), 1.93−1.85 (m, 2H), 1.75−1.67 (m, 2H), 1.65−1.58 (m, 2H),
1.40−1.32 (m, 2H). 13C NMR (126 MHz, MeOD) δ 167.2, 147.9,
138.8, 130.6, 130.5, 128.7, 126.7, 126.0, 124.0, 120.8, 103.3, 40.0, 35.5,
33.5, 26.0. MS (ESI) calcd for C17H17NO2 (m/z), 267.12; found,
268.14 [M + H]+.
2-(2-Hydroxypropyl)furo[2,3-c]quinoline 5-Oxide (7v). Solid (65
1
mg, 81%). H NMR (500 MHz, MeOD) δ 9.04 (d, J = 0.6 Hz, 1H),
2-(Cyclohexylmethyl)furo[2,3-c]quinoline 5-Oxide (7m). Solid
1
8.63−8.60 (m, 1H), 8.27−8.24 (m, 1H), 7.80−7.72 (m, 2H), 7.23 (d, J
= 0.7 Hz, 1H), 4.20−4.11 (m, 1H), 2.97 (ddd, J = 3.9, 2.6, 0.5 Hz,
2H), 1.21 (d, J = 6.2 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 164.5,
148.0, 138.8, 130.6, 130.5, 128.59, 126.8, 126.0, 124.0, 120.8, 104.6,
67.0, 39.2, 23.4. MS (ESI) calcd for C14H13NO3 (m/z), 243.10; found,
244.10 [M + H]+.
2-(2-Ethoxyethyl)furo[2,3-c]quinoline 5-Oxide (7w). Solid (30 mg,
70%). 1H NMR (500 MHz, MeOD) δ 9.15 (d, J = 0.7 Hz, 1H), 8.74−
8.71 (m, 1H), 8.39−8.35 (m, 1H), 7.91−7.84 (m, 2H), 7.34 (d, J = 0.9
Hz, 1H), 3.89 (t, J = 6.3 Hz, 2H), 3.58 (q, J = 7.0 Hz, 2H), 3.24−3.21
(m, 2H), 1.19 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, MeOD) δ
164.7, 147.9, 138.9, 130.6, 130.6, 128.6, 126.8, 126.0, 124.0, 120.8,
103.9, 68.6, 67.4, 30.4, 15.4. MS (ESI) calcd for C15H15NO3 (m/z),
257.10; found, 258.11 [M + H]+.
(160 mg, 84%). H NMR (500 MHz, CDCl3) δ 8.87 (s, 1H), 8.84
(dd, 1H), 8.09−8.05 (m, 1H), 7.76−7.69 (m, 2H), 6.86 (d, J = 0.7 Hz,
1H), 2.76 (d, J = 6.8 Hz, 2H), 1.90−1.52 (m, 6H), 1.33−1.14 (m,
3H), 1.08−0.97 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 162.9,
147.2, 138.7, 128.9, 128.5, 124.5, 124.3, 123.7, 122.8, 121.0, 102.3,
37.3, 36.6, 33.3, 29.9, 26.4, 26.2. MS (ESI) calcd for C18H19NO2 (m/
z), 281.14; found, 282.16 [M + H]+.
2-Phenylfuro[2,3-c]quinoline 5-Oxide (7n). White solid (73 mg,
68%). 1H NMR (500 MHz, CDCl3) δ 8.96 (d, J = 0.7 Hz, 1H), 8.89−
8.85 (m, 1H), 8.17 (ddd, J = 3.2, 2.2, 0.5 Hz, 1H), 7.94−7.90 (m, 2H),
7.80−7.74 (m, 2H), 7.53−7.49 (m, 2H), 7.47 (d, J = 0.9 Hz, 1H),
7.46−7.42 (m, 1H). 13C NMR (126 MHz, CDCl3) δ 159.6, 147.5,
139.1, 129.9, 129.3, 129.3, 129.2, 128.8, 125.3, 124.5, 124.4, 124.3,
122.9, 121.2, 100.2. MS (ESI) calcd for C17H11NO2 (m/z), 261.08;
found, 262.09 [M + H]+.
2-((1,3-Dioxoisoindolin-2-yl)methyl)furo[2,3-c]quinoline 5-Oxide
1
(7x). Solid (33 mg, 61%). H NMR (500 MHz, DMSO) δ 9.22 (s,
2-Benzylfuro[2,3-c]quinoline 5-Oxide (7o). White solid (96 mg
1H), 8.63−8.59 (m, 1H), 8.41−8.36 (m, 1H), 7.97−7.93 (m, 2H),
7.91−7.87 (m, 2H), 7.83−7.76 (m, 2H), 7.67 (d, J = 0.7 Hz, 1H), 5.06
(s, 2H). 13C NMR (126 MHz, DMSO) δ 167.2, 155.4, 147.4, 138.5,
134.8, 131.6, 129.3, 128.6, 124.9, 124.1, 123.5, 122.4, 121.6, 120.0,
104.4, 34.7. MS (ESI) calcd for C20H12N2O4 (m/z), 344.08; found,
345.09 [M + H]+.
1
90%). H NMR (500 MHz, CDCl3) δ 8.85 (d, J = 0.6 Hz, 1H), 8.83
(dd, 1H), 8.04−8.01 (m, 1H), 7.75−7.67 (m, 2H), 7.38 (ddd, J = 7.2,
4.4, 1.6 Hz, 2H), 7.35−7.30 (m, 3H), 6.82 (d, J = 0.9 Hz, 1H), 4.21 (s,
2H). 13C NMR (126 MHz, CDCl3) δ 162.0, 147.5, 138.9, 136.1, 129.1,
129.1, 129.0, 128.6, 127.4, 124.5, 124.2, 123.8, 122.8, 121.1, 102.7,
35.3. MS (ESI) calcd for C18H13NO2 (m/z), 275.10; found, 276.11 [M
+ H]+.
2-(Hydroxymethyl)furo[2,3-c]quinoline 5-Oxide(7p). Yellow solid
(77 mg, 79%). 1H NMR (500 MHz, MeOD) δ 9.15 (s, 1H), 8.72 (dd,
J = 7.9, 1.8 Hz, 1H), 8.38−8.31 (m, 1H), 7.91−7.82 (m, 2H), 7.43 (d,
J = 0.6 Hz, 1H), 4.82 (s, 2H). 13C NMR (126 MHz, MeOD) δ 165.1,
148.4, 139.0, 130.7, 130.7, 127.8, 127.0, 125.9, 124.2, 120.9, 103.9,
57.9. MS (ESI) calcd for C12H9NO3 (m/z), 215.10; found, 216.07 [M
+ H]+.
2-(2-(1,3-Dioxoisoindolin-2-yl)ethyl)furo[2,3-c]quinoline 5-Oxide
(7y). Solid (65 mg, 77%). 1H NMR (500 MHz, MeOD) δ 8.92 (d, J =
0.7 Hz, 1H), 8.70 (dd, J = 8.2, 1.5 Hz, 1H), 8.21−8.17 (m, 1H), 7.84−
7.77 (m, 4H), 7.77−7.73 (m, 2H), 7.19 (d, J = 0.8 Hz, 1H), 4.15 (t, J =
6.7 Hz, 2H), 3.35 (t, J = 7.1, 6.5 Hz, 2H). 13C NMR (126 MHz,
MeOD) δ 168.9, 161.8, 147.5, 138.4, 135.0, 134.9, 132.4, 130.0, 130.0,
127.3, 125.8, 125.3, 124.0, 123.9, 123.4, 120.5, 104.0, 36.6, 28.3. MS
(ESI) calcd for C21H14N2O4 (m/z), 358.10; found, 359.11 [M + H]+.
General Procedure for the Installation of the 4-Amino
Group. To a stirred solution of N-oxide (0.414 mmol) in CH2Cl2 was
added benzoylisocyanate (1.24 mmol). The resulting reaction mixture
was stirred at 55 °C for 2 h. After the completion of the reaction
(monitored by TLC), the solvent was evaporated. The residue was
redissolved in MeOH (4 mL), and NaOMe (2.07 mmol) was added
and refluxed for 4 h. The solvent was evaporated, and the crude
material was purified by flash chromatography using CH2Cl2/MeOH
as the eluent. For compounds 8x and 8y, after the reaction with
benzoylisocyanate (first step), the solvent was evaporated, and
ethylenediamine (2 mL) was added and stirred at 70 °C for 12 h.
2-(2-Hydroxyethyl)furo[2,3-c]quinoline 5-Oxide (7q). White solid
1
(75 mg, 78%). H NMR (500 MHz, MeOD) δ 9.09 (d, J = 0.5 Hz,
1H), 8.68 (dd, 1H), 8.31−8.27 (m, 1H), 7.88−7.78 (m, 2H), 7.29 (d, J
= 0.8 Hz, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.15 (td, J = 6.3, 0.7 Hz, 2H).
13C NMR (126 MHz, MeOD) δ 164.6, 147.9, 138.8, 130.6, 130.5,
128.5, 126.7, 125.9, 123.9, 120.8, 104.1, 60.5, 33.1. MS (ESI) calcd for
C13H11NO3 (m/z), 229.10; found, 230.09 [M + H]+.
2-(3-Hydroxypropyl)furo[2,3-c]quinoline 5-Oxide (7r). White solid
1
(75 mg, 88%). H NMR (500 MHz, MeOD) δ 9.13 (s, 1H), 8.75−
8.69 (m, 1H), 8.38−8.33 (m, 1H), 7.90−7.82 (m, 2H), 7.30 (d, J = 0.9
Hz, 1H), 3.69 (t, J = 6.2 Hz, 2H), 3.06 (t, 2H), 2.10−2.00 (m, 2H).
13C NMR (126 MHz, MeOD) δ 167.0, 148.0, 138.8, 130.6, 130.5,
128.7, 126.7, 126.0, 124.0, 120.8, 103.0, 61.8, 31.5, 26.0. MS (ESI)
calcd for C14H13NO3 (m/z), 243.10; found, 244.10 [M + H]+.
2-(4-Hydroxybutyl)furo[2,3-c]quinoline 5-Oxide (7s). Solid (60
1
Furo[2,3-c]quinolin-4-amine (8a). Solid (16 mg, 68%). H NMR
(500 MHz, CDCl3) δ 7.93 (ddd, J = 8.0, 1.5, 0.5 Hz, 1H), 7.81 (ddd, J
= 8.4, 1.1, 0.5 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.55 (ddd, J = 8.5, 7.0,
1.5 Hz, 1H), 7.38 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 7.20 (d, J = 2.0 Hz,
1H), 5.16 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 146.4, 145.2,
144.3, 139.5, 129.9, 127.8, 126.6, 123.4, 123.1, 120.5, 106.2. HRMS
(ESI) calcd for C11H8N2O (m/z), 184.0637; found, 185.0704 [M +
H]+.
2-Methylfuro[2,3-c]quinolin-4-amine (8b). Solid (35 mg, 70%).
1H NMR (500 MHz, CDCl3) δ 7.85 (ddd, J = 8.9, 5.0, 0.8 Hz, 2H),
7.55 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.36 (ddd, J = 8.1, 7.1, 1.2 Hz,
1
mg, 76%). H NMR (500 MHz, MeOD) δ 8.99 (s, 1H), 8.22−8.18
(m, 1H), 8.09 (dd, J = 8.4, 0.6 Hz, 1H), 7.70 (ddd, J = 8.4, 6.9, 1.6 Hz,
1H), 7.65 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.17 (d, J = 0.8 Hz, 1H), 3.63
(t, J = 6.4 Hz, 2H), 2.98 (t, 2H), 1.96−1.88 (m, 2H), 1.71−1.63 (m,
2H). 13C NMR (126 MHz, MeOD) δ 165.5, 149.8, 144.7, 136.6,
133.6, 129.5, 128.9, 128.0, 125.1, 124.3, 102.4, 62.4, 33.0, 29.2, 25.2.
I
dx.doi.org/10.1021/jm400694d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 6.81 (d, J = 1.0 Hz, 1H), 5.88 (s, 2H), 2.57 (d, J = 1.0 Hz, 3H).
13C NMR (126 MHz, CDCl3) δ 158.3, 144.9, 138.3, 132.0, 131.3,
129.7, 128.0, 125.3, 123.5, 123.1, 102.7, 14.5. HRMS (ESI) calcd for
C12H10N2O (m/z), 198.0793; found, 199.0859 [M + H]+.
9.6, 8.0. HRMS (ESI) calcd for C14H12N2O (m/z), 224.0950; found,
225.1016 [M + H]+.
2-Cyclopentylfuro[2,3-c]quinolin-4-amine (8k). Solid (40 mg,
1
68%). H NMR (500 MHz, CDCl3) δ 7.87 (dd, J = 8.0, 1.1 Hz,
2-Propylfuro[2,3-c]quinolin-4-amine (8c). Solid (38 mg, 78%). 1H
NMR (500 MHz, CDCl3) δ 7.88 (dd, J = 8.0, 1.2 Hz, 1H), 7.81−7.76
(m, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.34 (ddd, J = 8.0, 7.0,
1.1 Hz, 1H), 6.81 (t, J = 0.8 Hz, 1H), 5.12 (s, 2H), 2.89−2.81 (m,
2H), 1.88−1.80 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 161.7, 144.8, 144.1, 138.6, 131.4, 127.6, 126.5, 123.4,
122.8, 120.4, 101.8, 30.7, 21.3, 13.9. HRMS (ESI) calcd for
C14H14N2O (m/z), 226.1106; found, 227.1183 [M + H]+.
1H), 7.78 (dd, J = 8.4, 0.5 Hz, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz,
1H), 7.34 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.81 (d, J = 0.9 Hz, 1H), 5.13
(s, 2H), 3.35−3.27 (m, 1H), 2.20−2.11 (m, 2H), 1.89−1.82 (m, 3H),
1.79−1.70 (m, 3H). 13C NMR (126 MHz, CDCl3) δ 165.5, 144.8,
144.0, 138.5, 131.3, 127.6, 126.4, 123.4, 122.8, 120.4, 100.2, 39.3, 32.1,
29.9, 25.5. HRMS (ESI) calcd for C16H16N2O (m/z), 252.1263; found,
253.1338 [M + H]+.
2-(Cyclopentylmethyl)furo[2,3-c]quinolin-4-amine (8l). Solid (44
mg, 75%). 1H NMR (500 MHz, CDCl3) δ 7.88 (ddd, J = 7.9, 1.4, 0.4
Hz, 1H), 7.78 (dd, J = 8.4, 0.6 Hz, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz,
1H), 7.34 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.81 (s, 1H), 5.09 (s, 2H),
2.88−2.83 (m, 2H), 2.40−2.29 (m, 1H), 1.90−1.82 (m, 2H), 1.69
(qdd, J = 7.1, 6.7, 4.7 Hz, 2H), 1.64−1.54 (m, 2H), 1.36−1.26 (m,
2H). 13C NMR (126 MHz, CDCl3) δ 161.6, 144.8, 144.1, 138.6, 131.3,
127.6, 126.5, 123.4, 122.8, 120.4, 102.1, 38.9, 34.8, 32.7, 25.2. HRMS
(ESI) calcd for C17H18N2O (m/z), 266.1419; found, 267.1495 [M +
H]+.
1
2-Butylfuro[2,3-c]quinolin-4-amine (8d). Solid (39 mg, 80%). H
NMR (500 MHz, CDCl3) δ 7.87 (ddd, J = 8.0, 1.4, 0.4 Hz, 1H), 7.80−
7.76 (m, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.34 (ddd, J = 8.1,
7.0, 1.2 Hz, 1H), 6.80 (t, J = 0.8 Hz, 1H), 5.11 (s, 2H), 2.90−2.83 (m,
2H), 1.79 (ddd, J = 13.3, 8.5, 6.7 Hz, 2H), 1.51−1.40 (m, 2H), 0.98 (t,
J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 161.9, 144.8, 144.1,
138.6, 131.3, 127.6, 126.5, 123.4, 122.8, 120.4, 101.7, 30.0, 28.4, 22.4,
13.9. HRMS (ESI) calcd for C15H16N2O (m/z), 240.1263; found,
241.1349 [M + H]+.
1
2-Pentylfuro[2,3-c]quinolin-4-amine (8e). Solid (34 mg, 69%). H
2-(Cyclohexylmethyl)furo[2,3-c]quinolin-4-amine (8m). White
1
NMR (500 MHz, CDCl3) δ 7.88 (dd, J = 8.0, 1.1 Hz, 1H), 7.78 (dd, J
= 8.4, 0.5 Hz, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.34 (ddd, J =
8.1, 7.0, 1.2 Hz, 1H), 6.80 (s, 1H), 5.09 (s, 2H), 2.88−2.83 (m, 2H),
1.84−1.77 (m, 2H), 1.46−1.34 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 161.9, 144.8, 144.2, 138.6, 131.3, 127.6,
126.5, 123.4, 122.8, 120.4, 101.7, 31.5, 28.7, 27.6, 22.5, 14.1. HRMS
(ESI) calcd for C16H18N2O (m/z), 254.1419; found, 255.1496 [M +
H]+.
solid (75 mg, 71%). H NMR (500 MHz, CDCl3) δ 7.89−7.86 (m,
1H), 7.78 (dd, J = 8.4, 0.5 Hz, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz,
1H), 7.34 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 6.79 (s, 1H), 5.19 (s, 2H),
2.74 (d, J = 6.6 Hz, 2H), 1.81−1.65 (m, 6H), 1.32−1.14 (m, 4H), 1.04
(qd, J = 12.9, 2.8 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 160.8,
144.9, 144.1, 138.6, 131.3, 127.6, 126.4, 123.4, 122.8, 120.4, 102.7,
37.4, 36.6, 33.3, 26.4, 26.3. HRMS (ESI) calcd for C18H20N2O (m/z),
280.1576; found, 281.1656 [M + H]+.
2-Hexylfuro[2,3-c]quinolin-4-amine (8f). Yellow solid (60 mg
67%). 1H NMR (500 MHz, CDCl3) δ 7.90−7.86 (m, 1H), 7.79 (dd, J
= 8.4, 0.5 Hz, 1H), 7.52 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.34 (ddd, J =
8.1, 7.0, 1.2 Hz, 1H), 6.80 (s, 1H), 5.21 (s, 2H), 2.86 (t, 2H), 1.84−
1.76 (m, 2H), 1.47−1.39 (m, 2H), 1.38−1.29 (m, 4H), 0.91 (t, J = 7.1
Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 161.8, 144.8, 138.4, 131.2,
128.6, 127.5, 126.2, 123.3, 122.7, 120.2, 101.5, 31.5, 28.9, 28.6, 27.8,
22.6, 14.1. HRMS (ESI) calcd for C17H20N2O (m/z), 268.1576; found,
269.1658 [M + H]+.
2-Isobutylfuro[2,3-c]quinolin-4-amine (8g). Solid (55 mg, 70%).
1H NMR (500 MHz, CDCl3) δ 7.87 (ddd, J = 7.9, 1.4, 0.4 Hz, 1H),
7.80 (ddd, J = 8.5, 1.1, 0.5 Hz, 1H), 7.53 (ddd, J = 8.4, 7.0, 1.5 Hz,
1H), 7.34 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.81 (s, 1H), 5.36 (s, 2H),
2.73 (dd, J = 7.1, 0.6 Hz, 2H), 2.15 (dp, J = 13.6, 6.8 Hz, 1H), 1.02 (d,
J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 161.1, 144.9, 143.8,
138.6, 131.4, 127.6, 126.2, 123.4, 122.8, 120.2, 102.7, 37.9, 28.0, 22.6.
HRMS (ESI) calcd for C15H16N2O (m/z), 240.1263; found, 241.1337
[M + H]+.
2-Phenylfuro[2,3-c]quinolin-4-amine (8n). Solid (30 mg, 75%). 1H
NMR (500 MHz, CDCl3) δ 7.97 (ddd, J = 8.0, 1.4, 0.4 Hz, 1H), 7.94−
7.90 (m, 2H), 7.83−7.79 (m, 1H), 7.56 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H),
7.53−7.48 (m, 2H), 7.45−7.41 (m, 2H), 7.39 (ddd, J = 8.1, 7.0, 1.2
Hz, 1H), 5.23 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 157.8, 145.0,
144.4, 139.0, 131.7, 129.9, 129.5, 129.1, 127.9, 126.7, 125.3, 123.4,
123.1, 120.3, 100.8. HRMS (ESI) calcd for C17H12N2O (m/z),
260.0950; found, 261.1023 [M + H]+.
2-Benzylfuro[2,3-c]quinolin-4-amine (8o). Solid (45 mg, 65%). 1H
NMR (500 MHz, CDCl3) δ 7.86−7.82 (m, 1H), 7.77 (dd, J = 8.4, 0.6
Hz, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.40−7.35 (m, 2H),
7.35−7.28 (m, 4H), 6.79 (t, J = 0.9 Hz, 1H), 5.10 (s, 2H), 4.21 (s,
2H). 13C NMR (126 MHz, CDCl3) δ 159.8, 144.9, 144.2, 139.0, 136.7,
131.2, 129.1, 129.0, 127.7, 127.2, 126.5, 123.4, 122.9, 120.4, 103.2,
35.2. HRMS (ESI) calcd for C18H14N2O (m/z), 274.1106; found,
275.1182 [M + H]+.
(4-Aminofuro[2,3-c]quinolin-2-yl)methanol (8p). White solid (12
1
mg, 67%). H NMR (500 MHz, DMSO) δ 8.00 (dd, J = 7.9, 1.2 Hz,
2-(tert-Butyl)furo[2,3-c]quinolin-4-amine (8h). 1H NMR (500
MHz, CDCl3) δ 7.89 (ddd, J = 8.0, 1.5, 0.5 Hz, 1H), 7.79 (dd, J =
8.4, 0.5 Hz, 1H), 7.53 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.35 (ddd, J =
8.1, 7.0, 1.2 Hz, 1H), 6.80 (s, 1H), 5.27 (s, 2H), 1.45 (s, 9H). 13C
NMR (126 MHz, CDCl3) δ 169.8, 144.8, 143.6, 138.4, 131.3, 127.7,
126.1, 123.4, 123.0, 120.4, 99.0, 33.6, 29.2. HRMS (ESI) calcd for
C15H16N2O (m/z), 240.1263; found, 241.1342 [M + H]+.
1H), 7.59 (d, J = 7.9 Hz, 1H), 7.45 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.31
(s, 1H), 7.25 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.78 (s, 2H), 4.67 (s, 2H).
13C NMR (126 MHz, DMSO) δ 159.9, 146.1, 144.4, 138.6, 129.6,
127.1, 125.5, 123.5, 121.5, 119.4, 103.0, 56.4. HRMS (ESI) calcd for
C12H10N2O2 (m/z), 214.0742; found, 215.0805 [M + H]+.
2-(4-Aminofuro[2,3-c]quinolin-2-yl)ethanol (8q). White solid (20
1
mg, 65%). H NMR (500 MHz, CDCl3) δ 7.72 (d, J = 8.3 Hz, 1H),
2-Isopentylfuro[2,3-c]quinolin-4-amine (8i). Solid (35 mg, 70%).
1H NMR (500 MHz, CDCl3) δ 7.87 (dd, J = 8.0, 1.0 Hz, 1H), 7.79
(dd, J = 8.4, 0.5 Hz, 1H), 7.53 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.35
(ddd, J = 8.1, 7.1, 1.2 Hz, 1H), 6.81 (d, J = 0.8 Hz, 1H), 5.45−5.18 (m,
2H), 2.87 (t, 2H), 1.73−1.64 (m, 3H), 0.99 (d, J = 6.4 Hz, 6H). 13C
NMR (126 MHz, CDCl3) δ 162.5, 144.8, 138.3, 131.6, 128.8, 127.8,
126.0, 123.4, 123.0, 120.2, 101.6, 36.8, 27.8, 26.7, 22.5. HRMS (ESI)
calcd for C16H18N2O (m/z), 254.1419; found, 255.1496 [M + H]+.
2-Cyclopropylfuro[2,3-c]quinolin-4-amine (8j). Solid (28 mg,
7.65 (dd, J = 8.0, 1.1 Hz, 1H), 7.50 (ddd, J = 8.4, 5.9, 1.5 Hz, 1H),
7.30−7.26 (m, 1H), 6.79 (s, 1H), 5.28 (s, 2H), 4.10 (t, J = 6.0 Hz,
2H), 3.11 (t, J = 5.8 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 158.9,
144.6, 143.0, 138.3, 131.3, 127.8, 125.7, 123.4, 123.0, 119.8, 103.6,
60.5, 32.5. HRMS (ESI) calcd for C13H12N2O2 (m/z), 228.0899;
found, 229.0962 [M + H]+.
3-(4-Aminofuro[2,3-c]quinolin-2-yl)propan-1-ol (8r). Solid (70
1
mg, 75%). H NMR (500 MHz, CDCl3) δ 7.86 (dd, J = 8.0, 1.3
1
Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.52 (ddd, J = 8.4, 7.1, 1.5 Hz, 1H),
7.37−7.32 (m, 1H), 6.84 (s, 1H), 5.24 (s, 2H), 3.78 (t, J = 6.2 Hz,
2H), 3.00 (t, J = 7.5 Hz, 2H), 2.12−2.03 (m, 2H). 13C NMR (126
MHz, CDCl3) δ 161.1, 144.8, 143.8, 138.6, 131.4, 127.8, 126.2, 123.4,
123.0, 120.2, 102.1, 61.8, 30.8, 25.1. HRMS (ESI) calcd for
C14H14N2O2 (m/z), 242.1055; found, 243.1120 [M + H]+.
74%). H NMR (500 MHz, CDCl3) δ 7.85 (dd, J = 8.0, 1.1 Hz,
1H), 7.77 (dd, J = 8.4, 0.5 Hz, 1H), 7.52 (ddd, J = 8.4, 7.0, 1.5 Hz,
1H), 7.33 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 6.78−6.75 (m, 1H), 5.15−
5.03 (m, 2H), 2.14 (tt, J = 8.4, 5.1 Hz, 1H), 1.10 (tdd, J = 11.1, 7.0, 4.3
Hz, 2H), 1.05−0.99 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 162.7,
144.5, 143.9, 137.9, 131.5, 127.5, 126.3, 123.3, 122.7, 120.1, 99.9, 29.7,
J
dx.doi.org/10.1021/jm400694d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
4-(4-Aminofuro[2,3-c]quinolin-2-yl)butan-1-ol (8s). Solid (28 mg,
70%). 1H NMR (500 MHz, MeOD) δ 7.95−7.91 (m, 1H), 7.65 (dd, J
= 8.4, 0.5 Hz, 1H), 7.49 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.31 (ddd, J =
8.1, 7.1, 1.1 Hz, 1H), 7.01 (s, 1H), 3.63 (t, J = 6.5 Hz, 2H), 2.94 (t, J =
7.5 Hz, 2H), 1.95−1.87 (m, 2H), 1.70−1.63 (m, 2H). 13C NMR (126
MHz, MeOD) δ 163.9, 147.2, 144.2, 139.5, 133.0, 128.8, 125.5, 124.6,
123.7, 120.8, 102.8, 62.5, 33.0, 29.1, 25.3. HRMS (ESI) calcd for
C15H16N2O2 (m/z), 256.1212; found, 257.1282 [M + H]+.
2-(4-Aminofuro[2,3-c]quinolin-2-yl)propan-2-ol (8t). Solid (46
mg, 76%). 1H NMR (500 MHz, CDCl3) δ 7.77−7.71 (m, 2H),
7.52−7.47 (m, 1H), 7.29 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 6.86 (s, 1H),
1.72 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 165.5, 145.1, 143.3,
138.4, 130.8, 127.9, 125.8, 123.3, 123.1, 120.0, 100.3, 77.2, 69.2, 29.0.
HRMS (ESI) calcd for C14H14N2O2 (m/z), 242.1055; found, 243.1123
[M + H]+.
2-Butylfuro[3,2-c]quinoline (11b). Solid (258 mg, 69%). H NMR
(500 MHz, CDCl3) δ 9.13 (s, 1H), 8.25 (dd, J = 7.9, 1.6 Hz, 2H),
7.68−7.59 (m, 2H), 6.59 (t, J = 0.8 Hz, 1H), 2.94−2.87 (m, 2H), 1.81
(ddd, J = 15.2, 8.4, 6.7 Hz, 2H), 1.53−1.42 (m, 2H), 0.99 (t, J = 7.4
Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 160.5, 155.1, 145.4, 145.3,
129.9, 127.9, 126.8, 121.7, 119.9, 117.3, 101.3, 30.0, 28.3, 22.4, 13.9.
MS (ESI) calcd for C15H15NO (m/z), 225.11; found, 226.13 [M +
H]+.
1
2-Phenylfuro[3,2-c]quinoline (11c). Solid (68 mg, 75%). H NMR
(500 MHz, CDCl3) δ 9.18 (s, 1H), 8.38 (dd, J = 8.1, 1.2 Hz, 1H), 8.22
(d, J = 8.1 Hz, 1H), 7.98−7.93 (m, 2H), 7.74−7.64 (m, 2H), 7.51 (t, J
= 7.7 Hz, 2H), 7.44−7.39 (m, 1H), 7.21 (s, 1H). 13C NMR (126
MHz, CDCl3) δ 156.6, 155.3, 145.9, 145.5, 130.0, 129.9, 129.1, 129.1,
128.3, 127.1, 125.1, 122.05, 120.1, 117.3, 100.6. MS (ESI) calcd for
C17H11NO (m/z), 245.10; found, 246.09 [M + H]+.
Compounds 12a−12c were synthesized in a similar manner as
1-(4-Aminofuro[2,3-c]quinolin-2-yl)-3-methylbutan-1-ol (8u).
1
compound 7b.
Solid (35 mg, 77%). H NMR (500 MHz, MeOD) δ 8.00 (d, 1H),
2-Propylfuro[3,2-c]quinoline 5-oxide (12a). Solid (88 mg, 82%).
1H NMR (500 MHz, CDCl3) δ 8.87−8.82 (m, 1H), 8.79 (s, 1H),
8.25−8.20 (m, 1H), 7.76−7.70 (m, 2H), 6.51 (t, J = 0.9 Hz, 1H),
2.89−2.83 (m, 2H), 1.89−1.80 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 162.2, 147.4, 138.7, 130.7, 129.1, 128.8,
121.3, 120.9, 120.6, 117.9, 101.2, 30.5, 21.2, 13.9. MS (ESI) calcd for
C14H13NO2 (m/z), 227.10; found, 228.10 [M + H]+.
7.67 (d, J = 8.2 Hz, 1H), 7.53 (ddd, J = 8.4, 7.1, 1.4 Hz, 1H), 7.39−
7.33 (m, 1H), 7.24 (d, J = 2.6 Hz, 1H), 4.96 (t, J = 6.7 Hz, 1H), 1.92−
1.80 (m, 3H), 1.02 (dd, J = 6.2, 4.0 Hz, 6H). 13C NMR (126 MHz,
MeOD) δ 165.7, 147.3, 143.5, 132.8, 130.6, 129.2, 125.0, 124.7, 124.1,
120.6, 102.9, 66.9, 45.8, 25.7, 23.6, 22.3. HRMS MS (ESI) calcd for
C16H18N2O2 (m/z), 270.1368; found, 271.1468 [M + H]+.
1-(4-Aminofuro[2,3-c]quinolin-2-yl)propan-2-ol (8v). Solid (30
1
2-Butylfuro[3,2-c]quinoline 5-oxide (12b). Yellow solid (100 mg,
mg, 73%). H NMR (500 MHz, MeOD) δ 7.96 (dd, J = 8.2, 1.3 Hz,
1
79%). H NMR (500 MHz, CDCl3) δ 8.88−8.82 (m, 1H), 8.79 (s,
1H), 7.65 (dd, J = 8.4, 0.4 Hz, 1H), 7.50 (ddd, J = 8.5, 7.1, 1.5 Hz,
1H), 7.33 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.10 (s, 1H), 4.29−4.22 (m,
1H), 3.05−3.01 (m, 2H), 1.30 (d, J = 6.2 Hz, 3H). 13C NMR (126
MHz, MeOD) δ 161.3, 147.2, 144.0, 133.0, 130.4, 128.9, 125.3, 124.6,
123.8, 120.7, 104.4, 67.2, 39.0, 23.4. HRMS (ESI) calcd for
C14H14N2O2 (m/z), 242.1055; found, 243.1121 [M + H]+.
2-(2-Ethoxyethyl)furo[2,3-c]quinolin-4-amine (8w). Solid (15 mg,
71%). 1H NMR (500 MHz, MeOD) δ 8.01 (ddd, J = 8.0, 2.3, 1.2 Hz,
1H), 7.68 (d, J = 8.2 Hz, 1H), 7.57 (t, J = 7.3 Hz, 1H), 7.45−7.38 (m,
1H), 7.16 (s, 1H), 3.89 (t, J = 6.4 Hz, 2H), 3.58 (q, J = 7.0 Hz, 2H),
3.20 (dt, J = 6.5, 3.1 Hz, 2H), 1.20 (t, J = 7.0 Hz, 3H). 13C NMR (126
MHz, MeOD) δ 163.1, 146.51, 134.2, 133.1, 130.6, 129.7, 129.2,
125.0, 124.7, 123.3, 104.2, 68.9, 67.4, 30.3, 15.4. HRMS (ESI) calcd for
C15H16N2O2 (m/z), 256.1212; found, 257.1280 [M + H]+.
1H), 8.26−8.20 (m, 1H), 7.76−7.70 (m, 2H), 6.51 (t, J = 0.9 Hz, 1H),
2.91−2.85 (m, 2H), 1.83−1.76 (m, 2H), 1.51−1.43 (m, 2H), 0.99 (t, J
= 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 162.4, 147.4, 138.7,
130.7, 129.1, 128.8, 121.3, 121.0, 120.6, 117.9, 101.1, 29.9, 28.3, 22.4,
13.9. MS (ESI) calcd for C15H15NO2 (m/z), 241.11; found, 242.12 [M
+ H]+.
2-Phenylfuro[3,2-c]quinoline 5-oxide (12c). Solid (90 mg, 85%).
1H NMR (500 MHz, CDCl3) δ 8.90−8.86 (m, 2H), 8.37−8.33 (m,
1H), 7.94−7.90 (m, 2H), 7.78 (dd, J = 6.5, 3.3 Hz, 2H), 7.54−7.49
(m, 2H), 7.46−7.41 (m, 1H), 7.11 (s, 1H). 13C NMR (126 MHz,
CDCl3) δ 158.4, 147.6, 139.3, 130.7, 129.7, 129.4, 129.2, 129.2, 125.2,
121.4, 121.4, 120.8, 118.1, 100.0. MS (ESI) calcd for C17H11NO2 (m/
z), 261.10; found, 262.09 [M + H]+.
Compounds 13a−13c were synthesized in a similar manner as
2-(Aminomethyl)furo[2,3-c]quinolin-4-amine (8x). Solid (24 mg,
65%). 1H NMR (500 MHz, MeOD) δ 7.93 (dd, J = 8.0, 1.1 Hz, 1H),
7.65 (dd, J = 8.4, 0.4 Hz, 1H), 7.49 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.31
(ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.15 (s, 1H), 4.03 (s, 2H). 13C NMR
(126 MHz, MeOD) δ 162.7, 147.6, 145.0, 140.0, 132.4, 128.7, 126.0,
124.5, 123.6, 121.0, 102.9, 39.8. HRMS (ESI) calcd for C12H11N3O
(m/z), 213.0902; found, 214.0967 [M + H]+.
2-(2-Aminoethyl)furo[2,3-c]quinolin-4-amine (8y). Solid (25 mg,
66%). 1H NMR (500 MHz, MeOD) δ 7.94 (dd, J = 8.0, 1.1 Hz, 1H),
7.64 (dd, J = 8.4, 0.5 Hz, 1H), 7.48 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.30
(ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.07 (s, 1H), 3.14−3.02 (m, 4H). 13C
NMR (126 MHz, MeOD) δ 161.1, 147.4, 145.0, 140.0, 132.6, 128.6,
126.0, 124.5, 123.5, 120.9, 103.7, 40.9, 32.7. HRMS (ESI) calcd for
C13H13N3O (m/z), 227.1059; found, 228.1121 [M + H]+.
Compound 10 was synthesized in a similar manner as compound 5.
3-Iodoquinolin-4-ol (10). Solid (1.6 g). 1H NMR (500 MHz,
DMSO) δ 11.19 (s, 1H), 8.50 (s, 1H), 7.98−7.85 (m, 2H), 7.65−7.60
(m, 1H), 7.60−7.55 (m, 1H). 13C NMR (126 MHz, DMSO) δ 152.2,
142.6, 141.3, 130.9, 129.9, 129.2, 128.5, 126.6, 94.5. MS (ESI) calcd
for C9H6INO (m/z), 270.95; found, 271.96 [M + H]+.
compound 8a.
2-Propylfuro[3,2-c]quinolin-4-amine (13a). Solid (35 mg, 71%).
1H NMR (500 MHz, CDCl3) δ 8.05 (dd, 1H), 7.76 (d, J = 8.2 Hz,
1H), 7.52 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.34 (ddd, J = 8.1, 7.0, 1.1
Hz, 1H), 6.40 (t, J = 0.9 Hz, 1H), 5.10 (s, 2H), 2.86−2.81 (m, 2H),
1.87−1.78 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 159.1, 156.1, 151.9, 145.0, 128.2, 126.1, 122.9, 119.8, 114.7,
111.0, 99.7, 30.5, 21.4, 13.9. HRMS (ESI) calcd for C14H14N2O (m/z),
226.1106; found, 227.1177 [M + H]+.
2-Butylfuro[3,2-c]quinolin-4-amine (13b). White solid (55 mg,
1
69%). H NMR (500 MHz, CDCl3) δ 8.05 (ddd, J = 8.0, 1.5, 0.5 Hz,
1H), 7.79−7.73 (m, 1H), 7.52 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.34
(ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 6.39 (t, J = 0.9 Hz, 1H), 5.14 (s, 2H),
2.89−2.83 (m, 2H), 1.77 (ddd, J = 13.3, 8.5, 6.6 Hz, 2H), 1.50−1.41
(m, 2H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
159.3, 156.0, 151.9, 144.9, 128.3, 126.0, 123.0, 119.8, 114.7, 111.0,
99.6, 30.1, 28.2, 22.4, 13.9. HRMS (ESI) calcd for C15H16N2O (m/z),
240.1263; found, 241.1340 [M + H]+.
2-Phenylfuro[3,2-c]quinolin-4-amine (13c). Solid (33 mg, 67%).
1H NMR (500 MHz, MeOD) δ 8.93 (ddd, J = 8.0, 1.4, 0.4 Hz, 1H),
8.73 (dd, J = 8.4, 1.2 Hz, 2H), 8.47−8.44 (m, 1H), 8.35−8.31 (m,
1H), 8.31−8.27 (m, 2H), 8.21−8.12 (m, 3H). 13C NMR (126 MHz,
MeOD) δ 156.9, 156.5, 154.3, 145.9, 130.9, 129.7, 129.5, 129.4, 125.5,
125.6, 123.5, 120.6, 114.9, 112.9, 100.7, 49.0. HRMS (ESI) calcd for
C17H12N2O (m/z), 260.0950; found, 261.1027 [M + H]+.
Compounds 11a−11c were synthesized in a similar manner as
compound 6c.
1
2-Propylfuro[3,2-c]quinoline (11a). Solid (55 mg, 71%). H NMR
(500 MHz, CDCl3) δ 9.10 (s, 1H), 8.28−8.23 (m, 1H), 8.19 (dd, J =
8.0, 0.8 Hz, 1H), 7.67 (ddd, J = 8.5, 6.9, 1.6 Hz, 1H), 7.61 (ddd, J =
8.1, 7.0, 1.2 Hz, 1H), 6.60 (t, J = 0.9 Hz, 1H), 2.91−2.86 (m, 2H),
1.90−1.81 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 160.1, 155.1, 145.4, 145.2, 129.8, 127.8, 126.8, 121.6, 119.9,
117.3, 101.4, 30.5, 21.3, 13.9. MS (ESI) calcd for C14H13NO (m/z),
211.10; found, 212.11 [M + H]+.
Compound 15 was synthesized in a similar manner as compound 5.
1
4-Iodoisoquinolin-3-ol (15). Solid (1.4 g). H NMR (500 MHz,
DMSO) δ 8.84 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.76 (dd, J = 8.5, 0.6
Hz, 1H), 7.73−7.67 (m, 1H), 7.37 (ddd, J = 7.9, 6.8, 0.9 Hz, 1H). 13C
K
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NMR (126 MHz, DMSO) δ 159.8, 149.4, 140.9, 132.8, 128.8, 128.5,
123.8, 123.5. MS (ESI) calcd for C9H6INO (m/z), 270.95; found,
271.96 [M + H]+.
1H), 7.28−7.24 (m, 3H), 7.19 (dd, J = 10.3, 4.3 Hz, 2H), 4.38 (s, 2H),
2.91−2.86 (m, 2H), 1.80−1.72 (m, 2H), 1.44−1.35 (m, 2H), 0.93 (t, J
= 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 159.8, 148.3, 144.7,
138.9, 136.5, 136.3, 130.3, 130.1, 128.9, 128.0, 127.4, 126.9, 126.6,
126.5, 123.6, 113.9, 101.0, 30.6, 30.5, 26.4, 22.6, 13.9. MS (ESI) calcd
for C22H21NO (m/z), 315.20; found, 316.18 [M + H]+.
Compound 24 was synthesized in a similar manner as compound
8a.
1-Benzyl-2-butylfuro[2,3-c]quinolin-4-amine (24). Solid (31 mg,
75%). 1H NMR (500 MHz, CDCl3) δ 7.79−7.73 (m, 2H), 7.44 (ddd,
J = 8.4, 7.0, 1.4 Hz, 1H), 7.30−7.23 (m, 3H), 7.23−7.18 (m, 2H), 7.15
(ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 5.09 (s, 2H), 4.33 (s, 2H), 2.87−2.81
(m, 2H), 1.73 (ddd, J = 13.0, 8.5, 6.7 Hz, 2H), 1.39 (dq, J = 14.7, 7.4
Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
158.4, 144.9, 144.4, 139.1, 138.2, 130.0, 128.8, 128.0 (2C), 127.2 (2C),
126.6, 126.6, 123.4, 122.8, 121.0, 114.7, 30.7, 30.4, 26.3, 22.6, 13.9.
HRMS (ESI) calcd for C22H22N2O (m/z), 330.1732; found, 331.1808
[M + H]+.
Compound 16 was synthesized in a similar manner as compound
6c.
1
2-Butylfuro[2,3-c]isoquinoline (16). Solid (68 mg, 81%). H NMR
(500 MHz, CDCl3) δ 8.87 (s, 1H), 8.06 (dd, J = 8.5, 0.8 Hz, 2H), 7.74
(ddd, J = 8.4, 6.9, 1.2 Hz, 1H), 7.57−7.52 (m, 1H), 6.85 (t, J = 0.9 Hz,
1H), 2.93−2.87 (m, 2H), 1.82 (ddd, J = 13.3, 8.5, 6.7 Hz, 2H), 1.46
(dq, J = 14.8, 7.4 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 158.6, 158.3, 146.3, 131.3, 130.2, 128.8, 126.4, 125.2,
123.0, 114.4, 100.5, 29.9, 28.6, 22.4, 14.0. MS (ESI) calcd for
C15H15NO (m/z), 225.12; found, 226.12 [M + H]+.
Compound 17 was synthesized in a similar manner as compound
7b.
2-Butylfuro[2,3-c]isoquinoline 4-Oxide (17). Solid (20 mg, 74%).
1H NMR (500 MHz, MeOD) δ 8.94 (s, 1H), 8.24 (dd, J = 8.3, 0.9 Hz,
1H), 8.08 (d, J = 8.4 Hz, 1H), 7.83 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.70
(ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.30 (t, J = 0.9 Hz, 1H), 3.03−2.97 (m,
2H), 1.90−1.83 (m, 2H), 1.54−1.46 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, MeOD) δ 162.3, 134.6, 134.6, 131.8, 131.1,
130.5, 128.9, 128.1, 124.2, 121.7, 103.5, 30.8, 28.9, 23.3, 14.1. MS
(ESI) calcd for C15H15NO2 (m/z), 241.11; found, 242.11 [M + H]+.
Compound 18 was synthesized in a similar manner as compound
8a.
Compounds 27a,b were synthesized in a similar manner as
compound 7b.
1
2-Propylfuro[2,3-c]pyridine 6-oxide (27a). Oil (85 mg, 78%). H
NMR (500 MHz, CDCl3) δ 8.48 (s, 1H), 8.08 (dd, J = 6.7, 1.5 Hz,
1H), 7.30 (d, J = 6.7 Hz, 1H), 6.41 (d, J = 0.8 Hz, 1H), 2.75 (t, J = 7.5
Hz, 2H), 1.81−1.72 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 165.3, 152.0, 135.0, 128.5, 124.7, 115.8, 102.0, 30.5,
20.8, 13.8. MS (ESI) calcd for C10H11NO2 (m/z), 177.08; found,
178.08 [M + H]+.
2-Butylfuro[2,3-c]isoquinolin-5-amine (18). Solid (15 mg, 62%).
1H NMR (500 MHz, MeOD) δ 8.17−8.13 (m, 1H), 7.96 (ddd, J =
8.2, 1.1, 0.7 Hz, 1H), 7.69 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H), 7.43 (ddd, J
= 8.3, 6.9, 1.2 Hz, 1H), 6.74 (t, J = 0.9 Hz, 1H), 2.82−2.75 (m, 2H),
1.79−1.71 (m, 2H), 1.51−1.41 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, MeOD) δ 158.0, 156.0, 154.4, 134.4, 131.7, 125.9,
124.9, 124.1, 117.0, 106.1, 101.2, 31.3, 29.0, 23.3, 14.2. HRMS (ESI)
calcd for C15H16N2O (m/z), 240.1263; found, 241.1350 [M + H]+.
1-Bromo-2-butylfuro[2,3-c]quinoline (19). To a stirred solution of
2-butylfuro[2,3-c]quinoline 7d (100 mg, 0.44 mmol) in CH2Cl2 was
added bromine (80 μL, 1.55 mmol), and the mixture was stirred at
room temperature for 6 h. After the completion of the reaction
(monitored by TLC), the solvent was evaporated, and the crude
material was purified by flash chromatography using CH2Cl2/MeOH
1
2-Butylfuro[2,3-c]pyridine 6-Oxide (27b). Oil (80 mg, 73%). H
NMR (500 MHz, CDCl3) δ 8.52−8.47 (m, 1H), 8.10 (dd, J = 6.7, 1.5
Hz, 1H), 7.31 (d, J = 6.7 Hz, 1H), 6.42 (d, J = 0.9 Hz, 1H), 2.79 (t,
2H), 1.78−1.67 (m, 2H), 1.48−1.37 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, CDCl3) δ 165.5, 152.0, 135.1, 128.4, 124.8,
115.8, 101.9, 29.5, 28.3, 22.4, 13.9. MS (ESI) calcd for C11H13NO2
(m/z), 191.09; found, 192.10 [M + H]+.
Compounds 28a,b was synthesized in a similar manner as
compound 8a.
1
2-Propylfuro[2,3-c]pyridin-7-amine (28a). Oil (33 mg, 67%). H
NMR (500 MHz, CDCl3) δ 7.80 (d, J = 5.5 Hz, 1H), 6.85 (d, J = 5.5
Hz, 1H), 6.34 (t, J = 0.8 Hz, 1H), 4.67 (s, 2H), 2.79−2.71 (m, 2H),
1.82−1.74 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 161.8, 144.1, 140.5, 139.2, 135.3, 107.5, 102.4, 30.6, 21.1,
13.9. HRMS (ESI) calcd for C10H12N2O (m/z), 176.0950; found,
177.1067 [M + H]+.
1
to furnish 19 as a yellow solid (50 mg, 75%). H NMR (500 MHz,
CDCl3) δ 9.08 (s, 1H), 8.90 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.3 Hz,
1H), 7.71 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.68−7.63 (m, 1H), 2.98−
2.92 (m, 2H), 1.84−1.76 (m, 2H), 1.44 (dq, J = 14.8, 7.4 Hz, 2H),
0.98 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 159.4, 147.4,
144.5, 136.2, 130.1, 127.9, 127.7, 126.8, 122.9, 122.3, 94.0, 29.6, 26.6,
22.4, 13.9. MS (ESI) calcd for C15H14BrNO (m/z), 303.03; found,
304.04 [M + H]+.
2-Butylfuro[2,3-c]pyridin-7-amine (28b). Oil (35 mg, 71%). 1H
NMR (500 MHz, CDCl3) δ 7.80 (d, J = 5.5 Hz, 1H), 6.85 (d, J = 5.5
Hz, 1H), 6.33 (t, J = 0.8 Hz, 1H), 4.68 (s, 2H), 2.80−2.74 (m, 2H),
1.77−1.69 (m, 2H), 1.47−1.38 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 162.0, 144.0, 140.4, 139.2, 135.3, 107.5,
102.3, 29.8, 28.3, 22.4, 13.9. HRMS (ESI) calcd for C11H14N2O (m/z),
190.1106; found, 191.1231 [M + H]+.
Compound 21 was synthesized in a similar manner as compound
8a.
1-Bromo-2-butylfuro[2,3-c]quinolin-4-amine (21). Solid (21 mg,
72%). 1H NMR (500 MHz, MeOD) δ 8.67 (ddd, J = 8.2, 1.4, 0.5 Hz,
1H), 7.68 (ddd, J = 8.4, 1.1, 0.5 Hz, 1H), 7.53 (ddd, J = 8.5, 7.0, 1.5
Hz, 1H), 7.35 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 2.97 (t, J = 7.5 Hz, 2H),
1.86−1.78 (m, 2H), 1.50−1.41 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, MeOD) δ 159.7, 147.2, 145.3, 138.9, 129.1, 128.5,
126.3, 123.4, 123.1, 120.4, 95.0, 30.7, 27.2, 23.3, 14.1. HRMS (ESI)
calcd for C15H15BrN2O (m/z), 318.0368; found, 319.0429 [M + H]+.
1-Benzyl-2-butylfuro[2,3-c]quinoline (22). To a stirred solution of
19 (50 mg, 0.164 mmol) in 1,4-dioxane were added 2-benzyl-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane (54 mg, 0.246 mmol), Pd(dppf)Cl2 (8
mg, 0.009 mmol), Cs2CO3 (160 mg, 0.492 mmol), and 0.1 mL of
water. The resulting reaction mixture was stirred at 70 °C under a
nitrogen atmosphere for 12 h. After the completion of the reaction
(monitored by TLC), the reaction mixture was diluted with water and
extracted with ethylacetate (3 × 10 mL). The combined organic layer
was dried over Na2SO4 and concentrated under reduced pressure, and
the crude material was purified by flash chromatography using
Human TLR2, -3, -4, -5, -7, -8, and -9 Reporter-Gene Assays
(NF-κB Induction). The induction of NF-κB was quantified using
human TLR2, -3 ,-4, -5, -7, -8, and -9-specific HEK-Blue reporter-gene
assays as previously described by us.17,22,23 HEK293 cells stably
cotransfected with the appropriate hTLR, MD2, and secreted alkaline
phosphatase (sAP) were maintained in HEK-Blue Selection medium
containing zeocin and normocin. The stable expression of secreted
alkaline phosphatase (sAP) under the control of the NF-κB/AP-1
promoters is inducible by appropriate TLR agonists, and extracellular
sAP in the supernatant is proportional to the NF-κB induction. HEK-
Blue cells were incubated at a density of ∼105 cells/ml in a volume of
80 μL/well in 384-well, flat-bottomed, cell-culture-treated microtiter
plates until confluence was achieved, and they were subsequently
stimulated with graded concentrations of stimuli. sAP was assayed
spectrophotometrically using an alkaline phosphatase-specific chrom-
ogen (present in HEK-detection medium as supplied by the vendor) at
620 nm.
1
CH2Cl2/MeOH as the eluent to obtain 22 (24 mg, 92%). H NMR
Immunoassays for Cytokines. Fresh human peripheral blood
mononuclear cells (hPBMC) were isolated from human blood
obtained by venipuncture with informed consent and as per
(500 MHz, CDCl3) δ 9.11 (d, J = 5.7 Hz, 1H), 8.19−8.14 (m, 1H),
7.94 (dd, J = 8.3, 0.8 Hz, 1H), 7.61−7.54 (m, 1H), 7.44−7.37 (m,
L
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Journal of Medicinal Chemistry
Article
institutional guidelines on Ficoll-Hypaque gradients as described
elsewhere.59 Aliquots of PBMCs (105 cells in 100 μL/well) were
stimulated for 12 h with graded concentrations of the test compounds.
The supernatants were isolated by centrifugation and were assayed in
triplicate using analyte-specific multiplexed cytokine/chemokine bead-
array assays as reported by us previously.60 PBMC supernatants were
also analyzed for 41 chemokines and cytokines (EGF, Eotaxin, FGF-2,
Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-
10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1ra, IL-1α, IL-1β,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MCP-3,
MDC (CCL22), MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB,
RANTES, TGFα, TNF-α, TNF-β, VEGF, and sCD40L) using a
magnetic bead-based multiplexed assay kit (Milliplex MAP Human
Cytokine/Chemokine kit). Data were acquired and processed on a
MAGPIX instrument (EMD Millipore, Billerica, MA) with intraassay
coefficients of variation ranging from 4 to 8% for the 41 analytes.
Flow-Cytometric Immunostimulation Experiments. CD69
upregulation was determined by flow cytometry using protocols
published by us previously.17,18 Briefly, heparin-anticoagulated whole
blood samples were obtained by venipuncture from healthy human
volunteers with informed consent and as per guidelines approved by
the University of Kansas Human Subjects Experimentation Commit-
tee. Aliquots of whole human blood samples were stimulated with
graded concentrations of either 8d or 2 (used as a reference
compound) in a 6-well polystyrene plate and incubated at 37 °C in a
rotary (100 rpm) incubator for 16.5 h. Negative (endotoxin-free
water) controls were included in each experiment. Following
incubation, 200 μL aliquots of anticoagulated whole blood were
stained with 20 μL of fluorochrome-conjugated antibodies at 37 °C in
the dark for 30 min. For triple-color flow-cytometry experiments,
CD3-PE, CD56-APC, and CD69-PE-Cy7 were used to analyze CD69
activation in each of the main peripheral blood lymphocyte
populations: natural killer lymphocytes (NK cells; CD3−CD56+),
cytokine-induced killer phenotype (CIK cells; CD3+CD56+), nominal
B lymphocytes (CD3−CD56−), and nominal T lymphocytes
(CD3+CD56−). Following the staining, erythrocytes were lysed and
leukocytes were fixed in one step by mixing 200 μL of the samples in 4
mL of prewarmed Whole Blood Lyse/Fix Buffer (BD Biosciences, San
Jose, CA). After washing the cells twice at 200g for 8 min in saline, the
cells were transferred to a 96-well plate. Flow cytometry was
performed using a BD FACSArray instrument in the tricolor mode
(tricolor flow experiment) and the two-color mode (two-color flow
experiment) for the acquisition of 100 000 gated events. Postacqui-
sition analyses were performed using FlowJo v7.0 software (Treestar,
Ashland, OR). Compensation for spillover was computed for each
experiment on singly stained samples.
Transcriptomal Profiling of Human PBMCs. Detailed proce-
dures for transcriptomal profiling have been described by us
previously.17 Briefly, fresh human PBMC samples were stimulated
with 10 μg/mL of 8d and 2 for 2 h, and total RNA was extracted from
the treated and negative-control blood samples with a QIAamp RNA
Blood Mini Kit (Qiagen). Subsequently, 160 ng of each of the RNA
samples was used. The Human Genome GeneChip U133 plus 2.0
oligonucleotide array (Affymetrix, Santa Clara, CA) was employed.
Established standard protocols at the KU Genomics Facility were
performed for cRNA target preparation, array hybridization, washing,
staining, and image scanning. The microarray data was first subjected
to quality assessment using the Affymetrix GeneChip Operating
Software (GCOS). QC criteria included low background, low noise,
detection of the positive controls, and a 5′/3′ ratio of <3.0. To
facilitate the direct comparison of gene expression data between the
different samples, the GeneChip data were first subjected to
preprocessing. This step involved scaling (in GCOS) the data from
all chips to a target intensity value of 500 and further normalization
steps in GeneSpring GX (Agilent Technologies, Santa Clara, CA).
Prior to identifying the target genes, genes that were detected as
nonexpressed in all samples (i.e., those with absence calls) were
filtered out. To identify genes whose expression was changed by our
compounds, a fold-change threshold of 2.0 between the compound
treatment and the negative control was used.
Molecular Modeling and Induced-Fit Docking. We used
quantum mechanics/molecular mechanics (QM/MM) methods61,62
for induced-fit docking by calculating the quantum mechanical charges
for the ligand, whereas the macromolecule was handled using
conventional molecular mechanics force fields. The correct bond
orders were assigned, hydrogen atoms were added to the residues, and
formal partial charges were assigned to atoms using the OPLS all-atom
force field.63 The docking grid was generated using the cocrystallized
ligand as the grid center. Ligands were modeled in Schrodinger
̈
molecular modeling software (Schrodinger, New York, NY) and were
̈
minimized to a gradient of 0.001 kCal/MolÅ2. The QM charges for
the ligands were obtained from Jaguar (Schrodinger) using the 3-21G
̈
basis set with the BLYP density functional theory.64 Initial docking was
performed with Glide65,66 using a 0.5 van der Waals (vdW) radius
scaling factor for both the ligand and protein. This soft-docking
procedure was applied to generate diverse docking solutions, and the
top 20 poses for each ligand were retained. Finally, each ligand was
redocked into its corresponding structures, and the resulting
complexes were ranked according to GlideScore.65,66
ASSOCIATED CONTENT
* Supporting Information
■
S
1H and 13C spectra of compounds 6c−e, 7c−e, 8a, 8c−e, 11a,
11b, 12a, 12b, 13a, 13b, 16, 18, 19, 21, 24, 27a, 27b, 28a, and
28b and LC−MS analyses of compounds 8a, 8c−e, 13a, 13b,
18, 21, 24, 28a, and 28b. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 785-864-1610; Fax: 785-864-1961; E-mail: sdavid@ku.
■
edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by NIH/NIAID contract
HSN272200900033C.
■
ABBREVIATIONS:
■
APCs, antigen-presenting cells; CD69, cluster of differentiation
69; DCs, dendritic cells; EC50, half-maximal effective
concentration; ESI-TOF, electrospray ionization-time-of-flight;
G-CSF, granulocyte colony-stimulating factor; GRO, growth-
related oncogene; HEK, Human embryonic kidney; Ig,
immunoglobulin; IFN, interferon; MPL, monophosphoryl
lipid A; MIP, macrophage inflammatory protein; MHC,
major histocompatibility complex; NK, natural killer; NF-κB,
nuclear factor-κB; NOD, nucleotide oligomerization domain;
sAP, secreted alkaline phosphatase; SAR, structure activity
relationship; TNF-α, tumor necrosis factor-α; Th1, helper T
lymphocyte, type 1; Th2, helper T lymphocyte, type 2; Tregs,
T-regulatory cells; TGF, transforming growth factor; TLR, toll-
like receptor
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