Structure-activity relationships in human toll-like receptor 8-active 2,3-diamino-furo[2,3- c ]pyridines

Article abstract of DOI:10.1021/jm301066h

In our ongoing search toward identifying novel and synthetically simpler candidate vaccine adjuvants, we hypothesized that the imidazo[1,2-a]pyrazines, readily accessible via the Groebke-Blackburn-Bienaymé multicomponent reaction, would possess sufficient structural similarity with TLR7/8-agonistic imidazoquinolines. With pyridoxal as the aldehyde component, furo[2,3-c]pyridines, rather than the expected imidazo[1,2-a]pyridines, were obtained, which were characterized by NMR spectroscopy and crystallography. Several analogues were found to activate TLR8-dependent NF-κB signaling. In a focused library of furo[2,3-c]pyridines, a distinct SAR was observed with varying substituents at C2. In human PBMCs, none of the furo[2,3-c]pyridines showed any proinflammatory cytokine induction but upregulated several chemokine ligand genes. In immunization studies in rabbits, the most active compound showed prominent adjuvantic effects. The complete lack of proinflammatory cytokine induction coupled with strong adjuvantic activity of the novel furo[2,3-c]pyridines render this hitherto unknown chemotype an attractive class of compounds which are expected to be devoid of local or systemic reactogenicity.

Full text of DOI:10.1021/jm301066h

Article
pubs.acs.org/jmc
Structure−Activity Relationships in Human Toll-like Receptor
8‑Active 2,3-Diamino-furo[2,3‑c]pyridines
Deepak B. Salunke,† Euna Yoo,† Nikunj M. Shukla,† Rajalakshmi Balakrishna,† Subbalakshmi S. Malladi,†
Katelyn J. Serafin,† Victor W. Day,‡ Xinkun Wang,§ and Sunil A. David*,†
‡
§
†Department of Medicinal Chemistry Small-Molecule X-ray Crystallography Laboratory, Genomics Facility, University of Kansas,
Multidisciplinary Research Building, Room 320D, 2030 Becker Drive, Lawrence, Kansas 66047, United States
S
* Supporting Information
ABSTRACT: In our ongoing search toward identifying novel
and synthetically simpler candidate vaccine adjuvants, we
hypothesized that the imidazo[1,2-a]pyrazines, readily acces-
sible via the Groebke−Blackburn−Bienayme multicomponent
́
reaction, would possess sufficient structural similarity with
TLR7/8-agonistic imidazoquinolines. With pyridoxal as the
aldehyde component, furo[2,3-c]pyridines, rather than the
expected imidazo[1,2-a]pyridines, were obtained, which were
characterized by NMR spectroscopy and crystallography. Several analogues were found to activate TLR8-dependent NF-κB
signaling. In a focused library of furo[2,3-c]pyridines, a distinct SAR was observed with varying substituents at C2. In human
PBMCs, none of the furo[2,3-c]pyridines showed any proinflammatory cytokine induction but upregulated several chemokine
ligand genes. In immunization studies in rabbits, the most active compound showed prominent adjuvantic effects. The complete
lack of proinflammatory cytokine induction coupled with strong adjuvantic activity of the novel furo[2,3-c]pyridines render this
hitherto unknown chemotype an attractive class of compounds which are expected to be devoid of local or systemic
reactogenicity.
activation of TLRs by their cognate ligands leads to production
of inflammatory cytokines, and up-regulation of MHC
molecules and costimulatory signals in antigen-presenting
cells as well as activating natural killer cells (innate immune
response), which leads to the priming and amplification of T-
and B-cell effector functions (adaptive immune re-
sponses).14−17
INTRODUCTION
■
The innate and adaptive limbs of the immune system are
activated in a highly regulated and coordinated manner to
initiate host responses to invading pathogens. The innate
immune system utilizes germline-encoded pattern recognition
receptors (PRRs) to discern pathogen-associated molecular
patterns (PAMPs) that are distinct to the pathogen.1−3 PRRs
encompass diverse families of receptors4 that are secreted into
the extracellular environment (such as the collectins,5 ficolins,6
pentraxins,7 alarmins8), exist in the cytosol (examples of which
include the retinoic acid-inducible gene I-like receptors,9 and
the nucleotide-binding domain and leucine-rich repeat-
containing receptors10), or are present on membranes.
Important among the transmembrane PRRs include the Toll-
like receptors11 (TLRs), which are expressed either on the
plasma membrane or in the endolysosomal compartments.1 At
least 10 functional TLRs are encoded in the human genome,
each with an extracellular domain having leucine-rich repeats
and a cytosolic domain called the Toll/IL-1 receptor domain.12
The ligands for these receptors are highly conserved microbial
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).13 TLR1, -2, -4, -5, and -6
recognize extracellular stimuli, while TLR3, -7, -8, and -9
function within the endolysosomal compartment.12 The
The discovery of TLRs has not only served to greatly
accelerate our understanding of the interplay between the
innate and adaptive immune systems, but is also catalyzing
novel approaches to vaccine design and development. Several
synthetic compounds have been identified as ligands and
agonists of the TLRs, leading to detailed SAR studies on these
chemotypes. For instance, the S-[2,3-bis(palmitoyloxy)-(2RS)-
propyl]-R-cysteinyl-S-serine (PAM2CS) lipopeptides18 and
simpler analogues19,20 activate TLR2. Polyriboinosinic poly-
ribocytidylic acid (poly(I:C)) activates TLR3.21,22 Synthetic
lipid A23 and various monosaccharide lipid A analogues24,25
have been reported to activate immune cells by utilizing TLR4.
Imidazoquinolines such as imiquimod and resiquimod,26−28 2-
aminopyrimidines such as bromopirone,29,30 guanosine ana-
logues such as loxoribine,31 and 8-hydroxyadenine deriva-
tives32,33 are known to activate TLR7 and/or TLR8, while 2-
alkylthiazolo[4,5-c]quinolin-4-amine derivatives predominantly
activate TLR8 (Figure 1).34,35
Received: July 21, 2012
© XXXX American Chemical Society
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Scheme 1. Two Step One-Pot Synthetic Process
gene assays specific for human TLR-3, -7, -8, and -9; however,
compounds 26−29 obtained with pyridoxal as the aldehyde
component were found to specifically activate NF-κB signaling
in TLR8-transfected HEK293 cells. Detailed spectroscopic
analyses confirmed the formation of a hitherto unknown 2,3-
diaminofuro[2,3-c]pyridine skeleton via a noncanonical path-
way, which was unambiguously confirmed by single crystal X-
ray analysis. The TLR8-specific agonistic properties of this
novel and unexpected chemotype warranted a systematic SAR,
which is presented herein.
Figure 1. Representative TLR7/8-agonistic heterocyclic small
molecules.
Small molecule TLR7/8 activators constitute a small set of
compounds occupying a very small chemical space, and are
represented by substituted imidazo/thiazolo/oxazolo/
selenazolo[4,5-c]quinolines 1a−d, imidazo[4,5-c]pyridines 2,
and a few purine and pyrimidine derivatives (Figures 1 and 2).
RESULTS AND DISCUSSION
■
We have previously described extensive SAR on the 1,2-
disubstituted (1H-imidazo[4,5-c]quinoline-4-amines) class of
compounds27,28,40,41 (1a, Figure 2) as TLR7/8 agonists, and
their application in designing self-adjuvanting vaccine con-
structs.42 In our ongoing search toward identifying novel and
synthetically simpler candidate vaccine adjuvants, we hypothe-
sized that the imidazo[1,2-a]pyrazines 4 (Figure 2) would
possess sufficient structural similarity with the known small
molecule TLR7/8 ligands such as 1−3 (Figure 2). These
molecules are readily accessible in a one-pot, two-step process
́
using the Groebke−Blackburn−Bienayme multicomponent
reaction as a key step. An acid-catalyzed (HCl in dioxane),
microwave-mediated (400 W, 110 °C, 10 min) reaction using
2-amino-3-chloropyrazine (amidine component), isocyanocy-
clohexane (isonitrile component), and benzaldehyde (aldehyde
component) resulted, as expected, in 8-chloro-N-cyclohexyl-2-
phenylimidazo[1,2-a]pyrazin-3-amine (Scheme 1). Subsequent
microwave-mediated ipso-chloro displacement using ammo-
nium hydroxide was unsuccessful, but conventional heating in a
sealed tube (110 °C, 16 h) furnished the desired N3-cyclohexyl-
2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (5a; Scheme 1) in
30% yield over two steps. Using this one-pot process, a small
set of 8-aminoimidazo[1,2-a]pyrazines 5b−d was synthesized
by varying the aldehyde and isonitrile components (Scheme 1).
Simultaneously, a diverse test-library comprising 24 com-
pounds was also synthesized (Scheme 2) using two amidines
(2-aminopyridine and 2-aminopyrazine), three isonitriles (2-
isocyano-2-methylpropane, isocyanocyclohexane, and
(isocyanomethyl)benzene), and four aldehydes (benzaldehyde,
isonicotinaldehyde, salicylaldehyde, and pyridoxal). The syn-
theses of 6−23 (Scheme 2) proceeded smoothly. The typical
Groebke reaction, carried out at 100 °C for 20 min in CH3CN,
was found to be excessively harsh for reactions using pyridoxal
(24−29), leading to low yields and charring of reaction
mixtures. Reactions for this subset of compounds progressed
rapidly in 2 min under microwave conditions at 80 °C and 600
W power in CH3CN or MeOH. We noticed that only
Figure 2. TLR7/8 agonistic scaffolds. R1 is typically alkyl or O-alkyl, R2
is alkyl or benzyl, and R3 is usually alkyl, and OH in oxoadenines.
The identification of simpler molecules as TLR7/8 agonists
may pave the way for inexpensive vaccine constructs, and we
are therefore keenly interested in exploring alternative chemo-
types that are synthetically less complex. Unlike TLR2, TLR3,
TLR4,36 and TLR5,37 for which crystal structures are available
as complexes with their cognate ligands, a detailed structural
characterization of the mode of binding of TLR7 ligands is not
yet available to guide scaffold-hopping38,39 approaches. We
speculated that 3,8-diaminoimidazo[1,2-a]pyrazines 4 (Figure
2) may bear sufficient structural similarities to the known
TLR7/8 ligands (Figures 1 and 2). These molecules are, in
principle, readily accessible in two steps (one-pot synthetic
́
process) via the Groebke−Blackburn−Bienayme multicompo-
nent reaction,32,33 and we envisaged a rapid elaboration and
screening of a library of compounds for TLR7/8 agonistic
activities.
We began with the syntheses of small test-libraries of 3,8-
diamino-imidazo[1,2-a]pyrazines as well as 3-aminoimidazo-
[1,2-a]pyridine/pyrazines (Schemes 1 and 2). Most of these
compounds (5a−5d, 6−23) were inactive in NF-κB reporter
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Scheme 2. Twenty-four Membered Diverse Test-Library
compounds 24−29 (synthesized using pyridoxal) were
fluorescent on TLC under long-wave ultraviolet radiation.
The compounds were screened in NF-κB reporter gene
assays specific for human TLR-3, -4, -5, -7, -8, and -9. The 3,8-
diaminoimidazo[1,2-a]pyrazines 5a−d as well as 3-
aminoimidazo[1,2-a]pyridine/pyrazine library members 6−23
did not display any activity in these assays up to concentrations
of 250 μM (Supporting Information). However, compounds
26−29 (Scheme 2), obtained with the use of pyridoxal as the
aldehyde component, were found to specifically activate NF-κB
signaling in human TLR8-transfected HEK293 cells (Table 1,
Figure 3), but not human TLR-3, -4, -5, -7, and -9. The NMR
spectra of compounds 26−29 as well as their fluorescence
properties alerted us to the possibility of the formation of a new
chemical entity with a heterocyclic system other than the
classical imidazo[1,2-a]pyridine/pyrazines during the
ppm, whereas a pronounced downfield shift of this CH2 (up to
4.76 δ ppm) was observed in compounds 28 and 29. The 13C
NMR spectra of compounds 24−29 showed an unusual upfield
shift of one of the aromatic quaternary carbons in the region
(90−96 δ ppm). These observations suggested a different
heterocyclic system in 24−29. Initial efforts to crystallize these
molecules were unsuccessful. Pending continuing crystallization
efforts, we sought to elucidate the structures of these
compounds via alternate routes.
Three possible cyclization products appeared plausible in this
acid-catalyzed multicomponent reaction (Panel A in Scheme
3). As mentioned earlier, the NMR spectroscopic observations
for compounds 24−29 were not congruent with the 4-(3-
(cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-5-(hydroxy-
methyl)-2-methylpyridin-3-ol 32, the expected product via the
canonical Groebke mechanism (Path A, Panel A in Scheme 3).
In order to test whether the phenolic or benzylic hydroxyl
groups of pyridoxal may be involved in an alternate pathway of
cyclization, we carried out a reaction using a pyridoxal
derivative 34 with its phenolic hydroxyl protected with a
benzyl group (Scheme 4). This resulted in a product with a
spectral signature entirely consistent with the classical Groebke
product (5-(benzyloxy)-4-(3-(cyclohexylamino)imidazo[1,2-a]-
pyridin-2-yl)-6-methylpyridin-3-yl)methanol 35, indirectly also
́
Groebke−Blackburn−Bienayme multicomponent reaction. In
1H NMR spectra, the aliphatic CH of the cyclohexyl group in
compounds 8/9, 14/15, and 20/21 (Scheme 2) appeared in
the range of 3.0 to 3.1 δ ppm, whereas a pronounced downfield
shift of this CH proton (up to 3.85 δ ppm) was observed in
compounds 26 and 27. Similar observations were noted in
another set in which the aliphatic benzylic CH2 in compounds
10/11, 16/17, and 22/23 appeared in the range of 4.2 to 4.3 δ
C
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Table 1. EC50 Values of Compounds in Human TLR8-Specific Reporter Gene Assay
ruling out the possibility of cyclization involving the benzylic
hydroxyl group (Path B, Panel A in Scheme 3). We reasoned
that annulation via Path C ought to lead to a furo[2,3-
c]pyridine skeleton 26 and that if this were indeed the case, this
cyclization could proceed even if the amidine were to be
replaced with an aniline. We were gratified that a multi-
D
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alternate mechanism for this unusual cyclization route,
involving the pyridine ring system is proposed in Scheme 3
(Panel C).
After many unsuccessful attempts, a hydrochloride salt of
compound 28 was crystallized as multiply twinned bundles in
acetonitrile. A multidomain specimen of 28 was cut from one
bundle, which gave a set of diffracted intensities, permitting a
crystal structure solution (noncentrosymmetric, triclinic P1−
C1 space group with eight crystallographically independent
molecules in the asymmetric unit), but not a satisfactory
refinement (Figure 4). The structure of 28 unambiguously
confirmed the furo[2,3-c]pyridine chemotype. The structure of
compound 20 (obtained with salicylaldehyde, which also
possesses a phenolic OH; Scheme 2) was also elucidated,
which established the formation of a classic Groebke product 2-
(3-(cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)phenol (Fig-
ure 4). These observations clearly emphasize the significance
of the additional substituents of pyridoxal, directing the unique
cyclization route leading to the furo[2,3-c]pyridine scaffold.
Thus, our initial attempts toward the synthesis of a 24
membered imidazo[1,2-a]pyrazine/pyridine test-library unex-
pectedly resulted in the formation of densely substituted
furo[2,3-c]pyridines 24−29. Pyridoxal was found to be an
indispensable component for this cyclization reaction. Four of
the six compounds obtained (26−29, Scheme 2) were found to
be active in our primary screens using TLR8-transfected
HEK293 cells, while compounds 24 and 25, synthesized using
2-isocyano-2-methylpropane as one of the components, were
found to be inactive, warranting detailed structure based activity
relationship investigations for this new chemotype.
Among the active compounds 26−29, we observed that
compounds 26 and 28 (derived from 2-aminopyridine), were
more active than 27 and 29 (from 2-aminopyrazine; Table 1,
Figure 3). We therefore selected 2-aminopyridine and pyridoxal
as the invariant components and varied the isonitrile
component. We explored 13 different isonitriles (Scheme 6),
including linear aliphatic (as in 37a and 37b), branched
aliphatic (37c−e), linear aliphatic with silyl (37f), hetero-
aromatic ring (37g), ester (37h, 37i), and phosphate ester
(37j) termini, as well as aromatic substituents (37k−m).
Maximal activity was observed in 37b, with a pentylene
substituent on the C2 amine (Scheme 6, Figure 3). Diminishing
the chain length by one methylene unit (37a) decreased
activity, and potency was further attenuated in compounds with
α-branched substituents (37d). Compound 37n, with a free
NH2 at C2, obtained by N-dealkylation of the tert-octylamine
group43 of 37e with trifluoroacetic acid, as well as compounds
with aromatic substituents (37k−m) were devoid of TLR8-
stimulatory activity (Table 1). Thus, a distinct dependence of
the nature of the C2 amino substituent on the activity profiles
was observed in the furo[2,3-c]pyridines, with the C2-N-pentyl
(37b) and C2-N-(trimethylsilyl)methyl analogues (37f)
displaying dominant TLR8 agonism.
As mentioned earlier, our earlier efforts at unambiguously
confirming the structure of 28 did not allow for satisfactory
crystal structure refinement because of the intrinsic properties
of the crystal space group. Having synthesized 13 additional
furo[2,3-c]pyridines (37a−m) for the purposes of delineating
SAR, a parallel crystallization of these compounds was
attempted using various solvents. Suitable crystals of compound
37e were obtained as pale yellow crystals by slow evaporation
of a supersaturated solution of 37e in CH3CN/CH3OH
mixtures at room temperature. A single-domain specimen was
Figure 3. Dose−response profiles of TLR8 agonism by select 2,3-
diaminofuro[2,3-c]pyridines. Top: TLR8 agonism by compounds
derived from Schemes 2 and 5. Bottom: TLR8 agonism by compounds
derived from Schemes 6 and 7. Data points represent means and
standard deviations obtained on quadruplicates.
component reaction involving aniline, benzyl isonitrile, and
pyridoxal (Scheme 5) yielded the fluorescent compound 36,
whose 1H and 13C NMR spectra resembled those of
compounds 24−29. Interestingly, 36 was also found to be
weakly active (EC50 = 1.68 μM) in primary TLR8 screens
(Table 1, Figure 3). Our investigations thus far suggested the
formation of a hitherto unknown furo[2,3-c]pyridine structure,
exclusively when pyridoxal was used as the aldehyde
́
component in the Groebke−Blackburn−Bienayme multicom-
ponent reaction.
Although a definitive elucidation of the reaction mechanism
leading to the unexpected furo[2,3-c]pyridine was not an
immediate goal, understanding plausible mechanisms was of
interest and was probed in some detail. Formation of the
pyrano[3,4-c]pyridine 33 via nucleophilic attack of the benzylic
hydroxyl group and its subsequent rearrangement to the
furo[2,3-c]pyridine 26 (Path D, Panel A in Scheme 3) appeared
improbable. Salicylaldehyde, which lacks the bulky hydroxy-
methylene group, yielded the classical imidazo[1,2-a]pyridine
20 (confirmed by single crystal X-ray analysis, see below),
rather than the benzofuran derivative B.3 (Panel B in Scheme
3). We reasoned, therefore, that the benzylic hydroxyl in the
transition state 31 could assist cyclization via Path C (Panel A
in Scheme 3) due to steric reasons, apposing the phenolic
hydroxyl with the electrophilic carbon. In addition to the direct
formation of the furo[2,3-c]pyridine 26 via path C, a plausible
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Scheme 3. Possible Cyclization Pathways and Proposed Mechanism
Scheme 4
selected, and the X-ray diffraction data was collected. The
ORTEP diagram of 37e is shown in Figure 5, confirming the
non-Groebke furo[2,3-c]pyridine.
Previous mention was made that 36 (Scheme 5) was found
to be weakly active (EC50 = 1.68 μM, Table 1, Figure 3B)
relative to the lead compound 28, suggesting that the 2-
aminopyridine core could be substituted by anilines in this
multicomponent reaction. Having optimized the C2 group as
pentylamine (derived from 1-isocyanopentane, Scheme 6),
three more furo[2,3-c]pyridines were synthesized using aniline
38a, 3-fluoroaniline 38b, and 3-nitroaniline 38c in combination
with 1-isocyanopentane and pyridoxal (Scheme 7). The C2 N-
pentyl analogue 38a was found to be more active than the C2
N-benzyl analogue 36 (Figure 3A, Table 1). The nitro
derivative 38c was found to be as potent as the parent
compound 38a, whereas substantial gain in TLR8 activity was
noticed for the fluoro-substituted compound 38b in TLR8-
specific functional assays. However, these compounds exhibited
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Scheme 5
Scheme 6
Figure 4. Crystal structures of the salicylaldehyde-derived classic
Groebke product (imidazo[1,2-a]pyridine, 20), and a non-Groebke,
pyridoxal-derived furo[2,3-c]pyridine, 28.
a poorer dose−response profile (lower area-under the-curve,
Figure 3). It is pertinent to note that no stable product could be
obtained by the replacement of 2-aminopyridine with aliphatic
amines.
Several TLRs are thought to signal via ligand-induced
dimerization,44 as evident in the crystal structures of
TLR236,45 and TLR3.21 It is not yet understood, however,
how TLR7 and TLR8, whose endogenous ligands are single-
stranded viral RNA (ssRNA), recognize and transduce signals
upon engagement by small, nonpolymeric molecules such as
the imidazoquinolines26,46 and the oxoadenines.47,48 In order to
probe possible mechanisms of ligand recognition by TLR8, the
dimeric compound 39 was synthesized using 1,6-diisocyano-
hexane (Scheme 8); the activity of this analogue was
comparable in its TLR8-agonistic potency to the most active
compounds, 28, 37a, 37b, and 37f (Table 1).
Figure 5. Crystal structure (ORTEP view) of the non-Groebke
furo[2,3-c]pyridine, 37e, obtained with the use of pyridoxal as the
aldehyde component.
ence TLR8 agonist,34,35 which exhibited an EC50 of 1.32 μM
(Figure 3). In human PBMCs, only the reference thiazoloqui-
noline 40 but none of the furo[2,3-c]pyridines showed any
proinflammatory cytokine induction (Figure 6). We do not yet
know if the dissociation between TLR8-specific NF-κB
induction on the one hand and lack of cytokine induction on
the other is ascribable to a nonmyeloid differentiation primary
response gene 88 (MyD88)-independent mechanism.51,52
However, mindful of recent observations that proinflammatory
activity is not an absolute prerequisite for adjuvantic proper-
ties,53 and because we had previously observed potent NF-κB
We examined the cytokine-inducing properties49,50 of a
subset of compounds that were maximally active (28, 37a, 37b,
and 37f), all of which showed robust dose−response profiles in
primary TLR8-agonistic screens (Figure 3). We used 2-
propylthiazolo[4,5-c]quinolin-4-amine 40 (CL075) as a refer-
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MS using a Zorbax Eclipse Plus 4.6 mm × 150 mm, 5 μm analytical
reverse phase C18 column with either H2O−isopropanol or H2O−
CH3CN gradients, a diode-array detector operating in the 190−500
nm range (2 nm bandpass), and an Agilent ESI-TOF mass
spectrometer (integration on total ion intensity counts, with a mass
accuracy of 10 ppm) operating in the positive ion acquisition mode.
General Procedure for the Syntheses of Compounds 5a−f.
Synthesis of Compound 5a: N3-Cyclohexyl-2-phenylimidazo[1,2-
a]pyrazine-3,8-diamine. To a solution of 2-amino-3-chloropyrazine
(64 mg, 0.50 mmol) in anhydrous acetonitrile (1 mL) were added
benzaldehyde (60 μL, 0.60 mmol), 4 N HCl/dioxane (10 μL), and
cyclohexylisonitrile (74 μL, 0.60 mmol). The reaction mixture was
then heated under microwave conditions (400 W, 110 °C) in a sealed
vial for 20 min. The reaction mixture was cooled to room temperature;
ammonium hydroxide (NH3 content 28−30%, 0.5 mL) was added and
further heated at 110 °C in a sealed vial overnight. After the reaction
mixture was cooled to room temperature, the solvents were removed
and the residue was purified using column chromatography to obtain
Scheme 7
transactivation in a bisquinoline, 7-chloro-N-(4-(7-chloroqui-
nolin-4-ylamino)butyl) quinolin-4-amine 41 (RE-660)54 un-
accompanied by any proinflammatory cytokine induction, we
decided to examine two representative compounds (37b and
37f) in transcriptomal profiling experiments. Consistent with
the cytokine assays, there were no transcriptional signatures of
inflammation; however, both compounds upregulated several
chemokine ligand (both CXCL and CCL) genes (Supporting
Information). Although entirely bereft of any proinflammatory
activity, the bisquinoline compound 41 was found to be a
potent adjuvant which appears to be related to its functional
agonism at CCR1.54 Given some similarities in activity profiles
between the furo[2,3-c]pyridines and 41, we decided to
evaluate and compare the adjuvantic activity of 37b alongside
the reference compounds, 40 and 41. Rabbits were immunized
using bovine α-lactalbumin as a model subunit antigen.42 Anti-
α-lactalbumin IgG titers in immune sera clearly show an
adjuvantic effect of 37b, with a rise-in-titer values of >1000,
comparable to the adjuvantic activities of the reference
compounds, 40 and 41 (Figure 7). The complete lack of
proinflammatory cytokine induction coupled with strong
adjuvantic activity of the novel furo[2,3-c]pyridines render
this hitherto unknown chemotype an exceedingly attractive
class of compounds which are expected to be devoid of local or
systemic reactogenicity. Further structure−activity studies on
the furopyridines and related chemotypes are currently in
progress.
1
compound 5a (47 mg, 30%). H NMR (500 MHz, MeOD) δ 8.02
(dd, J = 8.2, 1.3 Hz, 2H), 7.64 (d, J = 4.8 Hz, 1H), 7.46 (dd, J = 10.9,
4.6 Hz, 2H), 7.39−7.28 (m, 1H), 7.21 (d, J = 4.8 Hz, 1H), 2.96−2.83
(m, 1H), 1.76 (d, J = 11.6 Hz, 2H), 1.71−1.62 (m, 2H), 1.57−1.51
(m, 1H), 1.31−1.19 (m, 2H), 1.19−1.03 (m, 3H). 13C NMR (126
MHz, MeOD) δ 151.14, 136.46, 135.17, 129.95, 129.55, 129.44,
128.67, 128.24, 128.11, 109.02, 57.87, 35.05, 26.86, 25.99. MS (ESI)
calcd for C18H21N5, m/z 307.1797, found 308.1923 (M + H)+.
Compounds 5b−d were synthesized similarly as compound 5a.
5b: 2-([1,1′-Biphenyl]-4-yl)-N3-cyclohexylimidazo[1,2-a]pyrazine-
3,8-diamine. (44 mg, 23%) 1H NMR (500 MHz, MeOD) δ 8.13 (d, J
= 8.2 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H), 7.71−7.67 (m, 2H), 7.64 (d, J
= 4.8 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.38−7.31 (m, 1H), 7.22 (d, J
= 4.8 Hz, 1H), 3.02−2.84 (m, 1H), 1.80 (d, J = 12.2 Hz, 2H), 1.69
(dd, J = 8.2, 5.3 Hz, 2H), 1.55 (s, 1H), 1.35−1.23 (m, 2H), 1.24−1.01
(m, 3H). 13C NMR (126 MHz, MeOD) δ 151.16, 141.92, 141.53,
136.10, 134.18, 130.07, 129.93, 129.56, 128.57, 128.45, 128.13, 128.02,
127.86, 109.03, 57.99, 35.12, 26.88, 26.03. MS (ESI) calcd for
C24H25N5, m/z 383.2110, found 384.2382 (M + H)+.
5c: N3-Benzyl-2-butylimidazo[1,2-a]pyrazine-3,8-diamine. (10
1
mg, 7%) H NMR (500 MHz, MeOD) δ 7.44 (d, J = 4.8 Hz, 1H),
7.29−7.17 (m, 5H), 7.12 (d, J = 4.8 Hz, 1H), 4.13 (s, 2H), 2.51−2.42
(m, 2H), 1.54−1.42 (m, 2H), 1.31 (dq, J = 14.8, 7.4 Hz, 2H), 0.91 (t, J
= 7.4 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 150.52, 141.10,
138.98, 130.14, 129.57, 129.49, 128.81, 128.40, 127.74, 108.93, 53.28,
32.76, 27.26, 23.73, 14.26. MS (ESI) calcd for C17H21N5, m/z
295.1797, found 296.1952 (M + H)+.
5d: 2-Butyl-N3-(4-methoxyphenyl)imidazo[1,2-a]pyrazine-3,8-di-
amine. (36 mg, 23%) 1H NMR (500 MHz, MeOD) δ 7.26 (d, J = 4.7
Hz, 1H), 7.16 (d, J = 4.7 Hz, 1H), 6.80−6.69 (m, 2H), 6.49−6.38 (m,
2H), 3.70 (s, 3H), 2.67 (t, J = 7.6 Hz, 2H), 1.73−1.62 (m, 2H), 1.41−
1.23 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, MeOD)
δ 154.65, 150.75, 142.09, 140.98, 129.90, 128.28, 124.98, 115.97,
115.50, 109.10, 56.07, 32.40, 27.41, 23.46, 14.15. MS (ESI) calcd for
C17H21N5O, m/z 311.1746, found 312.1905 (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 nitrogen atmosphere in
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, while thin-layer chromatography was carried out
on silica gel (200 μm) CCM precoated aluminum sheets. The purity of
all final compounds was confirmed to be greater than 95% by HPLC-
General Procedure for the Syntheses of Compounds 6−29.
Synthesis of Compound 6: N-(tert-Butyl)-2-phenylimidazo[1,2-
a]pyridin-3-amine. To a solution of 2-aminopyridine (24 mg, 0.25
mmol) in anhydrous acetonitrile were added benzaldehyde (28 μL,
0.28 mmol), 4 N HCl/dioxane (5 μL), and tert-butyl isonitrile (27 μL,
Scheme 8
H
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Figure 6. Dose−response profiles of proinflammatory cytokine induction in hPBMCs by compounds 28, 37a, 37b, and 37f. Representative data
from three independent experiments are presented.
0.24 mmol). The reaction mixture was then heated under microwave
conditions (400 W, 110 °C) in a sealed vial for 20 min. After the
reaction mixture was cooled to room temperature, the solvents were
removed and the residue was purified using column chromatography
to obtain compound 6 (44 mg, 70%). 1H NMR (400 MHz, CDCl3) δ
8.23 (dt, J = 6.9, 1.2 Hz, 1H), 7.90 (dt, J = 8.1, 1.6 Hz, 2H), 7.55 (dt, J
= 9.0, 1.0 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.31 (t, J = 7.4 Hz, 1H),
7.13 (ddd, J = 9.0, 6.6, 1.3 Hz, 1H), 6.77 (td, J = 6.8, 1.1 Hz, 1H), 3.12
(s, 1H), 1.04 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 142.15, 135.42,
135.40, 128.43, 128.32, 127.52, 124.17, 123.64, 117.48, 111.46, 101.91,
56.59, 30.43. MS (ESI) calcd for C17H19N3, m/z 265.1579, found
266.1664 (M + H)+.
30.45. MS (ESI) calcd for C16H18N4, m/z 266.1531, found 267.1588
(M + H)+.
8: N-Cyclohexyl-2-phenylimidazo[1,2-a]pyridin-3-amine. (64 mg,
1
86%) H NMR (500 MHz, CDCl3) δ 8.16 (d, J = 6.8 Hz, 1H), 8.05
(dd, J = 8.3, 1.2 Hz, 2H), 7.62 (d, J = 9.0 Hz, 1H), 7.45 (t, J = 7.8 Hz,
2H), 7.32 (t, J = 7.4 Hz, 1H), 7.17 (ddd, J = 8.9, 6.7, 1.2 Hz, 1H), 6.82
(td, J = 6.8, 0.9 Hz, 1H), 3.32 (s, 1H), 3.07−2.85 (m, 1H), 1.81 (d, J =
13.1 Hz, 2H), 1.68 (dd, J = 9.1, 3.6 Hz, 2H), 1.60−1.54 (m, 1H),
1.28−1.12 (m, 5H). 13C NMR (126 MHz, CDCl3) δ 141.02, 135.43,
133.45, 128.74, 127.74, 127.20, 125.17, 125.04, 123.07, 116.94, 112.30,
57.02, 34.27, 25.81, 24.94. MS (ESI) calcd for C19H21N3, m/z
291.1735, found 292.1832 (M + H)+.
9: N-Cyclohexyl-2-phenylimidazo[1,2-a]pyrazin-3-amine. (25 mg,
Compounds 7−29 were synthesized similarly as compound 6.
1
7: N-(tert-Butyl)-2-phenylimidazo[1,2-a]pyrazin-3-amine. (47 mg,
36%) H NMR (500 MHz, CDCl3) δ 8.99 (d, J = 1.4 Hz, 1H), 8.01
1
74%) H NMR (400 MHz, CDCl3) δ 9.00 (d, J = 1.4 Hz, 1H), 8.14
(ddd, J = 4.6, 3.2, 1.8 Hz, 3H), 7.85 (d, J = 4.6 Hz, 1H), 7.48 (t, J = 7.7
Hz, 2H), 7.38 (t, J = 7.4 Hz, 1H), 3.26 (s, 1H), 3.00 (m, 1H), 2.22 (s,
1H), 1.82 (dd, J = 6.6, 5.4 Hz, 2H), 1.70 (dd, J = 9.3, 3.3 Hz, 2H),
1.62−1.55 (m, 1H), 1.34−1.07 (m, 5H). 13C NMR (126 MHz,
CDCl3) δ 143.37, 139.08, 136.82, 133.62, 129.01, 128.90, 128.30,
(dd, J = 4.6, 1.5 Hz, 1H), 7.91 (dd, J = 8.3, 1.3 Hz, 2H), 7.86 (d, J =
4.6 Hz, 1H), 7.46 (t, J = 7.5 Hz, 2H), 7.37 (t, J = 7.4 Hz, 1H), 3.19 (s,
1H), 1.05 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 143.54, 142.40,
137.43, 134.38, 129.04, 128.66, 128.38, 128.33, 125.17, 116.49, 57.11,
I
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Hz, 2H), 8.07 (dt, J = 6.9, 1.2 Hz, 1H), 8.00 (dd, J = 4.6, 1.6 Hz, 2H),
7.54 (dt, J = 9.1, 1.0 Hz, 1H), 7.17 (ddd, J = 9.1, 6.6, 1.3 Hz, 1H), 6.81
(td, J = 6.8, 1.1 Hz, 1H), 3.11 (d, J = 3.9 Hz, 1H), 2.98 (td, J = 10.3,
4.1 Hz, 1H), 1.83 (d, J = 10.8 Hz, 2H), 1.71 (dd, J = 9.4, 3.3 Hz, 2H),
1.59 (dd, J = 7.3, 1.4 Hz, 1H), 1.32−1.09 (m, 5H). 13C NMR (126
MHz, CDCl3) δ 150.03, 142.26, 142.11, 133.78, 126.92, 124.92,
122.87, 121.21, 117.99, 112.31, 57.26, 34.42, 25.75, 24.97. MS (ESI)
calcd for C18H20N4, m/z 292.1688, found 293.1854 (M + H)+.
15: N-Cyclohexyl-2-(pyridin-4-yl)imidazo[1,2-a]pyrazin-3-amine.
1
(56 mg, 80%) H NMR (500 MHz, CDCl3) δ 9.01 (d, J = 1.5 Hz,
1H), 8.70 (dd, J = 4.5, 1.6 Hz, 2H), 8.03−7.93 (m, 3H), 7.88 (d, J =
4.6 Hz, 1H), 3.21 (d, J = 5.9 Hz, 1H), 3.06−2.97 (m, 1H), 1.84 (d, J =
10.5 Hz, 2H), 1.72 (dd, J = 9.7, 2.6 Hz, 2H), 1.63−1.58 (m, 1H),
1.31−1.14 (m, 5H). 13C NMR (126 MHz, CDCl3) δ 150.42, 144.32,
141.25, 137.17, 135.99, 129.45, 128.03, 121.37, 115.69, 57.35, 34.55,
25.62, 24.93. MS (ESI) calcd for C17H19N5, m/z 293.1640, found
294.1894 (M + H)+.
16: N-Benzyl-2-(pyridin-4-yl)imidazo[1,2-a]pyridin-3-amine. (54
1
mg, 75%) H NMR (500 MHz, CDCl3) δ 8.62 (dd, J = 4.6, 1.6 Hz,
2H), 7.94 (dt, J = 6.9, 1.1 Hz, 1H), 7.90 (dd, J = 4.6, 1.6 Hz, 2H), 7.55
(dt, J = 9.1, 1.0 Hz, 1H), 7.34−7.27 (m, 5H), 7.17 (ddd, J = 9.1, 6.6,
1.3 Hz, 1H), 6.76 (td, J = 6.8, 1.1 Hz, 1H), 4.22 (d, J = 6.1 Hz, 2H),
3.52 (t, J = 6.1 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 150.12,
142.11, 141.94, 138.64, 133.41, 128.98, 128.31, 128.06, 127.46, 125.05,
122.50, 121.13, 118.06, 112.42, 52.64. MS (ESI) calcd for C19H16N4,
m/z 300.1375, found 301.1599 (M + H)+.
Figure 7. Antibovine α-lactalbumin-specific IgG titers in rabbits
adjuvanted with 37b, 40, and 41 (n = 4, for each cohort). Box-plots of
ratios of immune/preimmune titers yielding absorbance values of 1.0
are shown for the individual samples. Means and medians of titers are
represented by and − symbols within the box, respectively, and the
× symbols indicate the 1% and 99% percentile values.
17: N-Benzyl-2-(pyridin-4-yl)imidazo[1,2-a]pyrazin-3-amine. (47
1
mg, 65%) H NMR (500 MHz, CDCl3) δ 9.01 (d, J = 1.4 Hz, 1H),
8.67 (dd, J = 4.5, 1.6 Hz, 2H), 7.87 (dd, J = 4.5, 1.6 Hz, 2H), 7.78 (dt,
J = 4.6, 3.0 Hz, 2H), 7.30 (dd, J = 5.1, 1.9 Hz, 3H), 7.24 (dd, J = 6.9,
2.6 Hz, 2H), 4.25 (d, J = 6.2 Hz, 2H), 3.66 (t, J = 6.2 Hz, 1H). 13C
NMR (126 MHz, CDCl3) δ 150.46, 144.33, 140.94, 138.23, 137.12,
135.70, 129.45, 129.14, 128.45, 128.32, 128.25, 121.34, 115.35, 52.59.
MS (ESI) calcd for C18H15N5, m/z 301.1327, found 302.1474 (M +
H)+.
□
127.42, 126.70, 115.73, 57.02, 34.41, 25.69, 24.91. MS (ESI) calcd for
C18H20N4, m/z 292.1688, found 293.1775 (M + H)+.
18: 2-(3-(tert-Butylamino)imidazo[1,2-a]pyridin-2-yl)phenol. (20
mg, 30%) 1H NMR (500 MHz, CDCl3) δ 12.47 (bs, 1H), 8.19 (d, J =
6.9 Hz, 1H), 8.12 (dd, J = 7.8, 1.6 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H),
7.24−7.17 (m, 2H), 7.07−6.97 (m, 1H), 6.95−6.87 (m, 1H), 6.83 (td,
J = 6.8, 0.9 Hz, 1H), 3.16 (bs, 1H), 1.16 (s, 9H). 13C NMR (126 MHz,
CDCl3) δ 157.07, 148.51, 140.76, 138.42, 134.51, 131.35, 129.43,
128.13, 124.82, 123.01, 118.69, 117.76, 116.96, 112.09, 109.94, 57.22,
30.50. MS (ESI) calcd for C17H19N3O, m/z 281.1528, found 282.1770
(M + H)+.
10: N-Benzyl-2-phenylimidazo[1,2-a]pyridin-3-amine. (62 mg,
86%) H NMR (500 MHz, CDCl3) δ 7.98 (ddt, J = 3.7, 3.0, 1.6
1
Hz, 3H), 7.57 (dt, J = 9.0, 1.0 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.39−
7.26 (m, 6H), 7.13 (ddd, J = 9.0, 6.7, 1.3 Hz, 1H), 6.74 (td, J = 6.8, 1.1
Hz, 1H), 4.20 (d, J = 6.1 Hz, 2H), 3.52 (t, J = 6.0 Hz, 1H), 2.45 (s,
1H). 13C NMR (126 MHz, CDCl3) δ 141.57, 139.06, 136.04, 134.13,
128.84, 128.30, 127.82, 127.65, 127.16, 125.77, 124.32, 122.50, 117.52,
111.94, 52.57. MS (ESI) calcd for C20H17N3, m/z 299.1422, found
300.1490 (M + H)+.
19: 2-(3-(tert-Butylamino)imidazo[1,2-a]pyrazin-2-yl)phenol. (40
11: N-Benzyl-2-phenylimidazo[1,2-a]pyrazin-3-amine. (45 mg,
1
1
mg, 59%) H NMR (500 MHz, CDCl3) δ 11.87 (s, 1H), 8.98 (d, J =
62%) H NMR (500 MHz, CDCl3) δ 8.98 (d, J = 1.3 Hz, 1H), 7.94
1.4 Hz, 1H), 8.16−8.08 (m, 2H), 7.92 (d, J = 4.6 Hz, 1H), 7.31−7.24
(m, 1H), 7.05 (dd, J = 8.2, 1.0 Hz, 1H), 6.93 (td, J = 7.9, 1.2 Hz, 1H),
3.22 (bs, 1H), 1.17 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 157.08,
142.79, 140.85, 135.93, 130.39, 129.72, 128.31, 123.47, 119.03, 118.04,
117.65, 115.85, 57.73, 30.54. MS (ESI) calcd for C16H18N4O, m/z
282.1481, found 283.1690 (M + H)+.
(dt, J = 8.1, 1.6 Hz, 2H), 7.82 (dd, J = 4.6, 1.4 Hz, 1H), 7.77 (d, J = 4.6
Hz, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.4 Hz, 1H), 7.34−7.27
(m, 5H), 4.23 (d, J = 2.4 Hz, 2H), 3.66 (s, 1H), 2.20 (s, 1H). 13C
NMR (126 MHz, CDCl3) δ 143.46, 138.76, 138.56, 136.79, 133.34,
129.06, 129.03, 128.99, 128.46, 128.24, 128.08, 127.42, 127.18, 115.38,
52.39. MS (ESI) calcd for C19H16N4, m/z 300.1375, found 301.1494
(M + H)+.
20: 2-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)phenol.
1
(30 mg, 41%) H NMR (500 MHz, CDCl3) δ 13.16 (s, 1H), 8.17
12: N-(tert-Butyl)-2-(pyridin-4-yl)imidazo[1,2-a]pyridin-3-amine.
1
(d, J = 6.8 Hz, 1H), 8.04 (dd, J = 7.8, 1.3 Hz, 1H), 7.50 (dt, J = 9.0, 0.9
Hz, 1H), 7.25−7.17 (m, 2H), 7.04 (dd, J = 8.2, 1.1 Hz, 1H), 6.95−
6.89 (m, 1H), 6.87 (td, J = 6.8, 1.0 Hz, 1H), 3.17−2.99 (m, 2H), 1.83
(d, J = 12.5 Hz, 2H), 1.72 (dd, J = 9.5, 3.1 Hz, 2H), 1.64−1.57 (m,
1H), 1.32 (dd, J = 22.7, 11.7 Hz, 2H), 1.26−1.13 (m, 3H). 13C NMR
(126 MHz, CDCl3) δ 157.67, 139.51, 136.00, 129.25, 126.41, 124.88,
123.55, 122.62, 118.82, 117.84, 117.40, 116.64, 112.46, 57.08, 34.19,
25.83, 24.96. MS (ESI) calcd for C19H21N3O, m/z 307.1685, found
308.1995 (M + H)+.
(50 mg, 78%) H NMR (500 MHz, CDCl3) δ 8.65 (d, J = 5.9 Hz,
2H), 8.20 (dt, J = 6.9, 1.0 Hz, 1H), 7.96 (dd, J = 4.7, 1.4 Hz, 2H), 7.56
(d, J = 9.1 Hz, 1H), 7.18 (ddd, J = 9.0, 6.6, 1.2 Hz, 1H), 6.81 (td, J =
6.8, 1.0 Hz, 1H), 3.07 (s, 1H), 1.10 (s, 9H). 13C NMR (126 MHz,
CDCl3) δ 149.69, 142.82, 142.39, 136.46, 124.86, 124.77, 123.38,
122.17, 117.68, 111.82, 56.78, 30.38. MS (ESI) calcd for C16H18N4, m/
z 266.1531, found 267.1747 (M + H)+.
13: N-(tert-Butyl)-2-(pyridin-4-yl)imidazo[1,2-a]pyrazin-3-amine.
1
(49 mg, 76%) H NMR (500 MHz, CDCl3) δ 9.02 (d, J = 1.4 Hz,
21: 2-(3-(Cyclohexylamino)imidazo[1,2-a]pyrazin-2-yl)phenol.
(54 mg, 73%) H NMR (500 MHz, CDCl3) δ 12.52 (bs, 1H), 8.96
1H), 8.69 (dd, J = 4.5, 1.6 Hz, 2H), 8.11 (dd, J = 4.7, 1.5 Hz, 1H), 7.94
(dd, J = 4.5, 1.6 Hz, 2H), 7.88 (d, J = 4.7 Hz, 1H), 3.12 (s, 1H), 1.10
(s, 9H). 13C NMR (126 MHz, CDCl3) δ 150.22, 144.25, 141.96,
139.25, 137.72, 129.39, 126.24, 122.42, 116.42, 57.46, 30.61. MS (ESI)
calcd for C15H17N5, m/z 267.1484, found 268.1705 (M + H)+.
14: N-Cyclohexyl-2-(pyridin-4-yl)imidazo[1,2-a]pyridin-3-amine.
1
(d, J = 1.4 Hz, 1H), 8.05 (ddd, J = 9.5, 6.2, 1.5 Hz, 2H), 7.94 (d, J =
4.6 Hz, 1H), 7.30−7.26 (m, 1H), 7.07 (dd, J = 8.2, 1.1 Hz, 1H), 6.94
(td, J = 8.0, 1.2 Hz, 1H), 3.31−2.99 (m, 2H), 1.83 (d, J = 12.3 Hz,
2H), 1.77−1.70 (m, 2H), 1.62 (dd, J = 7.5, 2.3 Hz, 1H), 1.33 (dd, J =
21.3, 10.5 Hz, 2H), 1.27−1.15 (m, 3H). 13C NMR (126 MHz, CDCl3)
1
(69 mg, 98%) H NMR (500 MHz, CDCl3) δ 8.64 (dd, J = 4.6, 1.6
J
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δ 157.70, 142.41, 138.53, 134.72, 130.19, 129.97, 126.50, 125.10,
119.13, 118.09, 116.60, 115.35, 57.17, 34.34, 25.69, 24.92. MS (ESI)
calcd for C18H20N4O, m/z 308.1637, found 309.1856 (M + H)+.
22: 2-(3-(Benzylamino)imidazo[1,2-a]pyridin-2-yl)phenol. (10
(t, J = 7.3 Hz, 1H), 4.64 (s, 2H), 4.61 (s, 2H), 2.61 (s, 3H). 13C NMR
(126 MHz, MeOD) δ 163.44, 156.59, 143.82, 143.17, 139.78, 139.64,
135.93, 134.61, 134.23, 134.16, 129.68, 128.54, 128.38, 125.19, 93.30,
59.41, 47.06, 14.34. MS (ESI) calcd for C20H19N5O2, m/z 361.1539,
found 362.1805 (M + H)+.
1
mg, 13%) H NMR (500 MHz, CDCl3) δ 13.02 (s, 1H), 8.06−7.95
(m, 2H), 7.51 (d, J = 9.0 Hz, 1H), 7.42−7.29 (m, 5H), 7.25−7.17 (m,
2H), 7.06 (dd, J = 8.2, 1.0 Hz, 1H), 6.95−6.89 (m, 1H), 6.80 (td, J =
6.8, 0.9 Hz, 1H), 4.24 (bs, 2H), 3.41 (s, 1H). 13C NMR (126 MHz,
CDCl3) δ 157.75, 139.49, 138.91, 135.58, 129.38, 128.96, 128.43,
127.97, 125.95, 124.95, 124.41, 122.19, 119.04, 117.88, 117.21, 116.74,
112.51, 52.43. MS (ESI) calcd for C20H17N3O, m/z 315.1372, found
316.1611 (M + H)+.
Synthesis of Compound 34: (2-(Benzylamino)-7-methyl-3-
(phenylamino)furo[2,3-c]pyridin-4-yl)methanol. To a solution of
aniline (45 μL, 0.5 mmol) in anhydrous methanol were added
pyridoxal hydrochloride (112 mg, 0.55 mmol), 4 N HCl/dioxane (10
μL), and benzyl isonitrile (68 μL, 0.55 mmol). The reaction mixture
was then heated under microwave conditions (600 W, 80 °C) in a
sealed vial for 2 min. After the reaction mixture was cooled to room
temperature, the solvents were removed and the residue was purified
using column chromatography to obtain compound 34 (84 mg, 47%).
1H NMR (500 MHz, MeOD) δ 7.88 (s, 1H), 7.34 (d, J = 7.8 Hz, 2H),
7.29 (t, J = 7.6 Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 7.09 (t, J = 7.9 Hz,
2H), 6.66 (td, J = 7.3, 0.9 Hz, 1H), 6.58−6.51 (m, 2H), 4.57 (s, 2H),
4.52 (s, 2H), 2.54 (s, 3H). 13C NMR (126 MHz, MeOD) δ 160.73,
150.14, 141.35, 141.10, 138.40, 137.10, 130.29, 129.51, 128.35, 128.23,
119.02, 114.15, 94.89, 60.27, 47.02, 16.66. MS (ESI) calcd for
C22H21N3O2, m/z 359.1634, found 360.1832 (M + H)+.
23: 2-(3-(Benzylamino)imidazo[1,2-a]pyrazin-2-yl)phenol. (43
1
mg, 57%) H NMR (500 MHz, CDCl3) δ 12.40 (bs, 1H), 8.95 (d,
J = 1.4 Hz, 1H), 8.02 (dd, J = 7.9, 1.6 Hz, 1H), 7.82 (d, J = 4.6 Hz,
1H), 7.78 (dd, J = 4.6, 1.4 Hz, 1H), 7.37−7.27 (m, 6H), 7.09 (dd, J =
8.3, 1.2 Hz, 1H), 6.94 (ddd, J = 7.9, 7.3, 1.2 Hz, 1H), 4.26 (d, J = 5.1
Hz, 2H), 3.53 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 157.70, 142.41,
138.44, 138.07, 134.64, 130.35, 129.95, 129.12, 128.39, 128.24, 126.21,
125.76, 119.36, 118.13, 116.33, 114.97, 52.35. MS (ESI) calcd for
C19H16N4O, m/z 316.1324, found 317.1545 (M + H)+.
Synthesis of Compound 35: 3-(Benzyloxy)-5-(hydroxymethyl)-2-
methylisonicotinaldehyde. To a solution of pyridoxalhydrochloride
(500 mg, 2.45 mmol) in anhydrous DMF were added potassium
carbonate (408 mg, 2.95 mmol) and benzyl bromide (0.35 mL, 2.95
mmol). The reaction mixture was stirred at room temperature
overnight. After the completion of the reaction, water (20 mL) was
added and the crude product obtained was extracted in ethyl acetate.
The organic layer was washed with water, dried over anhydrous
sodium sulfate, and evaporated. The residue was purified using column
24: (2-(tert-Butylamino)-7-methyl-3-(pyridin-2-ylamino)furo[2,3-
c]pyridin-4-yl)methanol. (13 mg, 17%) 1H NMR (400 MHz, MeOD)
δ 7.97 (d, J = 4.1 Hz, 1H), 7.87 (s, 1H), 7.59 (ddd, J = 8.7, 7.2, 1.8 Hz,
1H), 6.75 (ddd, J = 7.0, 5.2, 0.7 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 4.61
(s, 2H), 2.72 (s, 3H), 1.53 (s, 9H). 13C NMR (126 MHz, MeOD) δ
165.65, 159.92, 147.97, 143.43, 140.55, 139.89, 132.15, 131.86, 125.53,
115.57, 110.65, 96.72, 59.04, 55.33, 30.04, 13.08. MS (ESI) calcd for
C18H22N4O2, m/z 326.1743, found 327.1782 (M + H)+.
25: (2-(tert-Butylamino)-7-methyl-3-(pyrazin-2-ylamino)furo[2,3-
c]pyridin-4-yl)methanol. (14 mg, 18%) 1H NMR (500 MHz, MeOD)
δ 8.01 (d, J = 1.1 Hz, 1H), 7.88 (dd, J = 2.8, 1.4 Hz, 1H), 7.80 (s, 1H),
7.77 (d, J = 2.8 Hz, 1H), 4.56 (s, 2H), 2.65 (s, 3H), 1.43 (s, 9H). 13C
NMR (126 MHz, MeOD) δ 165.60, 156.50, 143.30, 143.00, 140.79,
134.76, 134.39, 132.05, 131.74, 125.39, 95.43, 58.99, 55.50, 29.98,
12.92. MS (ESI) calcd for C17H21N5O2, m/z 327.1695, found
328.1836 (M + H)+.
26: (2-(Cyclohexylamino)-7-methyl-3-(pyridin-2-ylamino)furo-
[2,3-c]pyridin-4-yl)methanol. (19 mg, 22%) 1H NMR (500 MHz,
MeOD) δ 8.07 (t, J = 7.4 Hz, 1H), 7.96 (s, 1H), 7.90 (d, J = 5.9 Hz,
1H), 7.29 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 6.7 Hz, 1H), 4.64 (s, 2H),
3.91−3.79 (m, 1H), 2.75 (s, 3H), 2.05 (d, J = 8.8 Hz, 2H), 1.87−1.77
(m, 2H), 1.68 (d, J = 13.1 Hz, 1H), 1.48−1.33 (m, 4H), 1.21 (dd, J =
12.1, 9.2 Hz, 1H). 13C NMR (126 MHz, MeOD) δ 164.86, 155.98,
145.65, 143.70, 142.10, 137.34, 133.72, 132.85, 124.81, 115.77, 115.45,
89.79, 59.50, 54.25, 34.33, 26.28, 26.16, 12.97. MS (ESI) calcd for
C20H24N4O2, m/z 352.1899, found 353.2023 (M + H)+.
27: (2-(Cyclohexylamino)-7-methyl-3-(pyrazin-2-ylamino)furo-
[2,3-c]pyridin-4-yl)methanol. (24 mg, 28%) 1H NMR (500 MHz,
MeOD) δ 8.10 (s, 1H), 7.97 (d, J = 1.4 Hz, 1H), 7.86 (s, 2H), 4.64 (s,
2H), 3.86−3.78 (m, 1H), 2.71 (s, 3H), 2.03−1.96 (m, 2H), 1.80 (dd, J
= 5.3, 3.1 Hz, 2H), 1.66 (d, J = 13.0 Hz, 1H), 1.38 (t, J = 9.6 Hz, 4H),
1.23−1.15 (m, 1H). 13C NMR (126 MHz, MeOD) δ 165.01, 156.57,
143.09, 142.98, 141.82, 134.49, 132.10, 131.50, 125.12, 94.07, 58.98,
53.94, 34.42, 26.31, 26.20, 12.80. MS (ESI) calcd for C19H23N5O2, m/z
353.1852, found 354.1902 (M + H)+.
28: (2-(Benzylamino)-7-methyl-3-(pyridin-2-ylamino)furo[2,3-c]-
pyridin-4-yl)methanol. (13 mg, 15%) 1H NMR (500 MHz, MeOD) δ
8.06−8.01 (m, 1H), 8.00 (s, 1H), 7.89 (d, J = 5.9 Hz, 1H), 7.44−7.39
(m, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.3 Hz, 1H), 7.23 (d, J =
8.6 Hz, 1H), 7.05 (t, J = 6.7 Hz, 1H), 4.76 (s, 2H), 4.65 (s, 2H), 2.77
(s, 3H). 13C NMR (126 MHz, MeOD) δ 165.45, 156.13, 145.31,
143.75, 142.15, 138.61, 138.18, 133.67, 133.22, 130.23, 130.00, 129.91,
128.97, 128.77, 125.32, 115.82, 115.01, 90.66, 59.42, 47.33, 12.98. MS
(ESI) calcd for C21H20N4O2, m/z 360.1586, found 361.1830 (M +
H)+.
29: (2-(Benzylamino)-7-methyl-3-(pyrazin-2-ylamino)furo[2,3-c]-
pyridin-4-yl)methanol. (15 mg, 16%) 1H NMR (500 MHz, MeOD) δ
7.99 (s, 1H), 7.94 (dd, J = 2.8, 1.5 Hz, 1H), 7.87 (s, 1H), 7.81 (d, J =
2.8 Hz, 1H), 7.37−7.32 (m, 2H), 7.29 (dd, J = 10.3, 5.0 Hz, 2H), 7.21
1
chromatography to obtain compound 35 (100 mg, 15%). H NMR
(500 MHz, CDCl3) δ 8.05 (s, 1H), 7.45−7.32 (m, 5H), 6.64 (d, J =
1.7 Hz, 1H), 5.34 (d, J = 11.2 Hz, 1H), 5.22 (d, J = 12.7 Hz, 1H), 5.19
(d, J = 11.2 Hz, 1H), 4.99 (d, J = 12.7 Hz, 1H), 2.50 (s, 3H). 13C
NMR (126 MHz, CDCl3) δ 150.88, 148.63, 136.84, 136.17, 135.53,
135.42, 128.80, 128.47, 127.74, 100.10, 74.13, 69.95, 19.38. MS (ESI)
calcd for C15H15NO3, m/z 257.1052, found 258.1279 (M + H)+.
Synthesis of Compound 36: (5-(Benzyloxy)-4-(3-
(cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)-6-methylpyridin-3-
yl)methanol. To a solution of 2-aminopyridine (24 mg, 0.25 mmol) in
anhydrous acetonitrile were added 3-(benzyloxy)-5-(hydroxymethyl)-
2-methylisonicotinaldehyde 34 (68 mg, 0.28 mmol), 4 N HCl/dioxane
(5 μL), and cyclohexylisonitrile (30 μL, 0.24 mmol). The reaction
mixture was then heated under microwave conditions (400 W, 110
°C) in a sealed vial for 20 min. After the reaction mixture was cooled
to room temperature, solvents were removed and the residue was
purified using column chromatography to obtain compound 36 (19
1
mg, 17%). H NMR (500 MHz, CDCl3) δ 8.41 (s, 1H), 8.07 (d, J =
6.9 Hz, 1H), 7.54 (d, J = 9.1 Hz, 1H), 7.26−7.15 (m, 4H), 7.02−6.97
(m, 2H), 6.90 (td, J = 6.8, 1.0 Hz, 1H), 4.59 (bs, 1H), 4.43 (s, 2H),
4.36 (bs, 1H), 3.88 (d, J = 7.9 Hz, 1H), 2.61 (bs, 1H), 2.58 (s, 3H),
1.85−1.75 (m, 1H), 1.70−1.65 (m, 1H), 1.50−1.40 (m, 2H), 1.37−
1.28 (m, 1H), 1.22−1.05 (s, 2H), 1.05−0.85 (m, 2H), 0.60 (bs, 1H).
13C NMR (126 MHz, CDCl3) δ 152.95, 150.53, 145.20, 141.29,
135.58, 129.62, 129.25, 128.76, 128.58, 128.57, 128.40, 126.28, 125.05,
123.16, 117.47, 112.53, 76.73, 61.64, 56.36, 34.32, 33.74, 25.60, 25.03,
24.53, 19.58. MS (ESI) calcd for C27H30N4O2, m/z 442.2369, found
443.2410 (M + H)+.
General Procedure for the Syntheses of Compounds 37a−
m. Synthesis of Compound 37a: (2-(Butylamino)-7-methyl-3-
(pyridin-2-ylamino)furo[2,3-c]pyridin-4-yl)methanol. To a solution
of 2-aminopyridine (24 mg, 0.25 mmol) in anhydrous acetonitrile
were added pyridoxal hydrochloride (56 mg, 0.28 mmol), 4 N HCl/
dioxane (5 μL), and n-butyl isonitrile (25 μL, 0.24 mmol). The
reaction mixture was then heated under microwave conditions (600
W, 80 °C) in a sealed vial for 2 min. After the reaction mixture was
cooled to room temperature, solvents were removed and the residue
was purified using column chromatography to obtain compound 37a
1
(32 mg, 41%). H NMR (500 MHz, MeOD) δ 8.06 (t, J = 8.0 Hz,
1H), 7.96 (s, 1H), 7.90 (d, J = 5.8 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H),
K
dx.doi.org/10.1021/jm301066h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.07 (t, J = 6.6 Hz, 1H), 4.65 (s, 2H), 3.56 (t, J = 7.1 Hz, 2H), 2.75 (s,
3H), 1.75−1.61 (m, 2H), 1.49−1.35 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, MeOD) δ 165.68, 155.98, 145.61, 143.66,
142.08, 137.58, 133.67, 132.87, 124.84, 115.82, 115.31, 90.01, 59.50,
43.64, 32.79, 20.93, 13.99, 12.96. MS (ESI) calcd for C18H22N4O2, m/z
326.1743, found 327.1925 (M + H)+.
115.98, 115.25, 91.02, 62.95, 59.42, 44.55, 14.48, 13.03. MS (ESI)
calcd for C18H20N4O4, m/z 356.1485, found 357.1738 (M + H)+.
37i: tert-Butyl 3-((4-(Hydroxymethyl)-7-methyl-3-(pyridin-2-
ylamino)furo[2,3-c]pyridin-2-yl)amino)propanoate. (20 mg, 21%)
1H NMR (500 MHz, MeOD) δ 8.08 (t, J = 8.0 Hz, 1H), 8.00 (s, 1H),
7.91 (d, J = 5.8 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 6.7 Hz,
1H), 4.66 (s, 2H), 3.80 (t, J = 6.4 Hz, 2H), 2.78 (s, 3H), 2.67 (t, J =
6.7 Hz, 2H), 1.42 (s, 9H). 13C NMR (126 MHz, MeOD) δ 171.99,
165.49, 155.86, 145.77, 143.76, 142.08, 137.49, 133.79, 133.30, 125.17,
115.86, 115.36, 90.25, 82.28, 59.49, 39.67, 36.03, 28.31, 13.02. MS
(ESI) calcd for C21H26N4O4, m/z 398.1954, found 399.2128 (M +
H)+.
Compounds 37b−m were synthesized similarly as compound 37a.
37b: (7-Methyl-2-(pentylamino)-3-(pyridin-2-ylamino)furo[2,3-
c]pyridin-4-yl)methanol. (27 mg, 33%) 1H NMR (500 MHz,
MeOD) δ 8.06 (t, J = 8.0 Hz, 1H), 7.96 (s, 1H), 7.91 (d, J = 5.9
Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 6.6 Hz, 1H), 4.65 (s,
2H), 3.56 (t, J = 7.1 Hz, 2H), 2.75 (s, 3H), 1.75−1.64 (m, 2H), 1.38
(d, J = 3.6 Hz, 4H), 0.92 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz,
MeOD) δ 165.68, 156.04, 145.55, 143.66, 142.09, 137.74, 133.66,
132.85, 124.85, 115.82, 115.25, 90.09, 59.49, 43.90, 30.42, 29.98,
23.31, 14.32, 12.95. MS (ESI) calcd for C19H24N4O2, m/z 340.1899,
found 341.2058 (M + H)+.
37c: (2-(Isopropylamino)-7-methyl-3-(pyridin-2-ylamino)furo-
[2,3-c]pyridin-4-yl)methanol. (20 mg, 27%) 1H NMR (500 MHz,
MeOD) δ 8.05 (t, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.90 (d, J = 5.9 Hz,
1H), 7.26 (d, J = 8.6 Hz, 1H), 7.05 (t, J = 6.7 Hz, 1H), 4.63 (s, 2H),
4.25 (dt, J = 13.0, 6.5 Hz, 1H), 2.74 (s, 3H), 1.33 (d, J = 6.5 Hz, 6H).
13C NMR (126 MHz, MeOD) δ 164.96, 156.16, 145.41, 143.72,
142.06, 137.79, 133.63, 132.82, 124.86, 115.77, 115.24, 90.04, 59.48,
47.16, 23.12, 12.94. MS (ESI) calcd for C17H20N4O2, m/z 312.1586,
found 313.1740 (M + H)+.
37d: (7-Methyl-2-(pentan-2-ylamino)-3-(pyridin-2-ylamino)furo-
[2,3-c]pyridin-4-yl)methanol. (34 mg, 42%) 1H NMR (500 MHz,
MeOD) δ 7.99 (t, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.83 (d, J = 5.2 Hz,
1H), 7.20 (d, J = 7.3 Hz, 1H), 7.00 (t, J = 6.7 Hz, 1H), 4.56 (s, 2H),
4.03 (dq, J = 12.9, 6.5 Hz, 1H), 2.66 (s, 3H), 1.60−1.41 (m, 2H),
1.38−1.26 (m, 2H), 1.23 (d, J = 6.6 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, MeOD) δ 165.14, 155.98, 145.72, 143.66,
37j: Diethyl (((4-(Hydroxymethyl)-7-methyl-3-(pyridin-2-
ylamino)furo[2,3-c]pyridin-2-yl)amino)methyl)phosphonate. (13
1
mg, 13%) H NMR (500 MHz, MeOD) δ 8.08−8.00 (m, 2H), 7.95
(d, J = 5.7 Hz, 1H), 7.23 (d, J = 4.9 Hz, 1H), 7.06 (t, J = 6.5 Hz, 1H),
4.69 (s, 2H), 4.24−4.16 (m, 4H), 4.09 (d, J = 10.6 Hz, 2H), 2.81 (s,
3H), 1.33 (t, J = 7.0 Hz, 6H). 13C NMR (126 MHz, MeOD) δ 164.98,
156.41, 145.04, 143.68, 142.06, 139.12, 133.83, 133.71, 125.96, 115.97,
114.67, 91.93, 64.56 (d, J = 6.9 Hz), 59.37, 39.02 (d, J = 158.9 Hz),
16.78 (d, J = 5.6 Hz), 13.09. MS (ESI) calcd for C19H25N4O5P, m/z
420.1563, found 421.1672 (M + H)+.
37k: (2-((4-Methoxyphenyl)amino)-7-methyl-3-(pyridin-2-
1
ylamino)furo[2,3-c]pyridin-4-yl)methanol. (13 mg, 14%) H NMR
(500 MHz, MeOD) δ 7.99−7.87 (m, 2H), 7.62 (t, J = 7.8 Hz, 1H),
7.33 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 6.77 (t, J = 6.3 Hz,
2H), 4.66 (s, 2H), 3.77 (s, 3H), 2.71 (s, 3H). 13C NMR (126 MHz,
MeOD) δ 162.79, 159.21, 158.92, 146.57, 143.34, 141.31, 140.61,
132.84, 132.22, 130.80, 126.51, 124.33, 115.64, 115.43, 111.46, 96.53,
62.30, 58.97, 55.97, 13.06. MS (ESI) calcd for C21H20N4O3, m/z
376.1535, found 377.1694 (M + H)+.
37l: (2-((2-Chloro-6-methylphenyl)amino)-7-methyl-3-(pyridin-2-
1
ylamino)furo[2,3-c]pyridin-4-yl)methanol. (27 mg, 28%) H NMR
(500 MHz, MeOD) δ 8.10 (s, 1H), 7.90 (dd, J = 20.4, 6.6 Hz, 2H),
7.28−7.15 (m, 3H), 7.03 (t, J = 6.6 Hz, 1H), 6.95 (s, 1H), 4.70 (s,
2H), 2.75 (s, 3H), 2.32 (s, 3H). 13C NMR (126 MHz, MeOD) δ
162.57, 155.17, 145.80, 144.21, 142.68, 139.93, 136.98, 134.45, 133.96,
133.78, 132.14, 130.74, 128.65, 126.80, 115.93, 114.82, 91.45, 59.30,
18.71, 13.11. MS (ESI) calcd for C21H19ClN4O2, m/z 394.1197, found
395.1349 (M + H)+.
142.17, 137.38, 133.77, 132.84, 124.79, 115.82, 115.41, 89.73, 59.52,
51.18, 39.97, 21.53, 20.53, 14.15, 12.94. MS (ESI) calcd for
C19H24N4O2, m/z 340.1899, found 341.2077 (M + H)+.
37e: (7-Methyl-3-(pyridin-2-ylamino)-2-((2,4,4-trimethylpentan-
2-yl)amino)furo[2,3-c]pyridin-4-yl)methanol. (33 mg, 36%) 1H
NMR (500 MHz, MeOD) δ 8.08 (t, J = 8.0 Hz, 1H), 7.99 (s, 1H),
7.90 (s, 1H), 7.27 (s, 1H), 7.08 (dd, J = 7.0, 6.4 Hz, 1H), 4.63 (s, 2H),
2.78 (s, 3H), 1.95 (d, J = 12.9 Hz, 2H), 1.60 (s, 6H), 1.00 (s, 9H). 13C
NMR (126 MHz, MeOD) δ 165.37, 156.14, 145.64, 143.96, 141.40,
137.05, 133.87, 132.93, 124.92, 115.77, 115.62, 90.82, 59.88, 59.51,
53.31, 32.62, 31.82, 30.73, 13.06. MS (ESI) calcd for C22H30N4O2, m/z
382.2369, found 383.2541 (M + H)+.
37f: (7-Methyl-3-(pyridin-2-ylamino)-2-(((trimethylsilyl)methyl)-
amino)furo[2,3-c]pyridin-4-yl)methanol. (24 mg, 28%) 1H NMR
(500 MHz, MeOD) δ 8.03 (t, J = 8.0 Hz, 1H), 7.91−7.90 (m, 2H),
7.24 (d, J = 8.7 Hz, 1H), 7.04 (t, J = 6.6 Hz, 1H), 4.62 (s, 2H), 3.11 (s,
2H), 2.72 (s, 3H), 0.12 (s, 9H). 13C NMR (126 MHz, MeOD) δ
165.66, 156.25, 145.27, 143.32, 142.02, 138.19, 133.57, 132.16, 124.28,
115.77, 115.00, 90.25, 59.52, 34.64, 12.94, −2.60. MS (ESI) calcd for
C18H24N4O2Si, m/z 356.1669, found 357.1815 (M + H)+.
37g: (7-Methyl-2-((2-morpholinoethyl)amino)-3-(pyridin-2-
ylamino)furo[2,3-c]pyridin-4-yl)methanol. (9 mg, 10%) 1H NMR
(500 MHz, MeOD) δ 8.03 (s, 2H), 7.93 (d, J = 5.8 Hz, 1H), 7.27 (d, J
= 7.3 Hz, 1H), 7.05 (t, J = 6.5 Hz, 1H), 4.66 (s, 2H), 4.06−3.95 (m,
6H), 3.54 (bs, 2H), 3.42 (bs, 4H), 2.80 (s, 3H). 13C NMR (126 MHz,
MeOD) δ 165.51, 156.26, 145.10, 143.92, 141.95, 138.27, 134.09,
133.62, 126.01, 115.97, 115.07, 90.35, 64.80, 59.39, 57.37, 53.45,
37.84, 13.21. MS (ESI) calcd for C20H25N5O3, m/z 383.1957, found
384.2132 (M + H)+.
37h: Ethyl 2-((4-(Hydroxymethyl)-7-methyl-3-(pyridin-2-
ylamino)furo[2,3-c]pyridin-2-yl)amino)acetate. (21 mg, 25%) 1H
NMR (500 MHz, MeOD) δ 8.07 (t, J = 8.0 Hz, 1H), 8.03 (s, 1H),
7.91 (d, J = 6.1 Hz, 1H), 7.29 (d, J = 8.9 Hz, 1H), 7.08 (t, J = 6.7 Hz,
1H), 4.66 (s, 2H), 4.36 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 2.74 (s, 3H),
1.25 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 170.68,
165.42, 155.75, 145.81, 143.87, 142.15, 137.71, 133.94, 133.89, 125.93,
37m: (S)-(7-Methyl-2-((1-phenylethyl)amino)-3-(pyridin-2-
1
ylamino)furo[2,3-c]pyridin-4-yl)methanol. (28 mg, 31%) H NMR
(500 MHz, MeOD) δ 7.92 (s, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.90−7.81
(m, 1H), 7.38 (d, J = 7.6 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 7.21 (t, J =
7.2 Hz, 1H), 7.05 (d, J = 6.1 Hz, 1H), 6.94 (t, J = 6.4 Hz, 1H), 5.20 (q,
J = 6.8 Hz, 1H), 4.58 (s, 2H), 2.68 (s, 3H), 1.60 (d, J = 6.9 Hz, 3H).
13C NMR (126 MHz, MeOD) δ 164.81, 157.13, 144.72, 143.97,
143.61, 142.11, 140.61, 133.20, 132.80, 129.92, 128.71, 126.98, 125.52,
115.70, 113.93, 91.91, 59.24, 54.49, 23.31, 12.88. MS (ESI) calcd for
C22H22N4O2, m/z 374.1743, found 375.1931 (M + H)+.
Synthesis of Compound (37n): (2-Amino-7-methyl-3-(pyridin-2-
ylamino)furo[2,3-c]pyridin-4-yl)methanol. The solution of com-
pound 37e (95 mg, 0.25 mmol) in TFA/CH2Cl2 (50%, 4 mL) was
stirred at room temperature for 6 h. The solvents were removed, and
the residue obtained was column purified to afford compound 37n (56
1
mg, 84%). H NMR (500 MHz, MeOD) δ 7.91−7.86 (m, 2H), 7.81
(t, J = 7.9 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 6.87 (t, J = 6.5 Hz, 1H),
4.61 (s, 2H), 2.66 (s, 3H). 13C NMR (126 MHz, MeOD) δ 167.39,
157.68, 143.36, 143.14, 142.12, 132.82, 132.44, 125.47, 115.59, 113.10,
92.54, 59.18, 12.80. MS (ESI) calcd for C14H14N4O2, m/z 270.1117,
found 271.1310 (M + H)+.
Compounds 38a−c were synthesized similarly as compound 34.
38a: (7-Methyl-2-(pentylamino)-3-(phenylamino)furo[2,3-c]-
pyridin-4-yl)methanol. (117 mg, 70%) 1H NMR (500 MHz,
MeOD) δ 7.85 (s, 1H), 7.14 (dd, J = 8.6, 7.4 Hz, 2H), 6.71 (tt, J =
7.4, 1.0 Hz, 1H), 6.60 (dd, J = 8.6, 1.0 Hz, 2H), 4.65−4.59 (m, 2H),
3.51 (t, J = 7.1 Hz, 2H), 2.68 (s, 3H), 1.72−1.57 (m, 2H), 1.41−1.28
(m, 4H), 0.95−0.84 (m, 3H). 13C NMR (126 MHz, MeOD) δ 165.54,
149.56, 143.22, 141.35, 133.46, 132.44, 130.44, 125.33, 119.45, 114.08,
L
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96.74, 59.31, 43.42, 31.00, 29.94, 23.33, 14.34, 13.48. MS (ESI) calcd
for C20H25N3O2, m/z 339.1947, found 340.2087 (M + H)+.
38b: (3-((3-Fluorophenyl)amino)-7-methyl-2-(pentylamino)furo-
[2,3-c]pyridin-4-yl)methanol. (70 mg, 82%) 1H NMR (500 MHz,
MeOD) δ 7.81 (s, 1H), 7.06 (td, J = 8.2, 6.7 Hz, 1H), 6.42−6.32 (m,
2H), 6.23 (d, J = 11.5 Hz, 1H), 4.60 (s, 2H), 3.46 (t, J = 7.1 Hz, 2H),
2.65 (s, 3H), 1.59 (p, J = 7.1 Hz, 2H), 1.31−1.23 (m, 4H), 0.83 (t, J =
7.0 Hz, 3H). 13C NMR (126 MHz, MeOD) 166.34, 165.62 (d, J =
242.1 Hz), 151.55 (d, J = 10.4 Hz), 142.79, 142.04, 131.84 (d, J = 10.1
Hz), 131.65, 131.28, 125.42, 110.01, 105.58 (d, J = 21.8 Hz), 100.74
(d, J = 25.8 Hz), 96.47, 58.95, 43.43, 30.83, 29.93, 23.33, 14.35, 12.79.
MS (ESI) calcd for C20H24FN3O2, m/z 357.1853, found 358.2013 (M
+ H)+.
38c: (7-Methyl-3-((3-nitrophenyl)amino)-2-(pentylamino)furo-
[2,3-c]pyridin-4-yl)methanol. (34 mg, 37%) 1H NMR (500 MHz,
MeOD) δ 7.90 (s, 1H), 7.49 (dd, J = 8.1, 2.2 Hz, 1H), 7.34 (s, 1H),
7.31 (t, J = 8.1 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 4.54 (s, 2H), 3.41 (t,
J = 7.0 Hz, 2H), 2.56 (s, 3H), 1.65−1.52 (m, 2H), 1.30 (dd, J = 8.5,
4.8 Hz, 4H), 0.86 (t, J = 6.5 Hz, 3H). 13C NMR (126 MHz, MeOD) δ
160.86, 151.59, 150.89, 144.93, 141.84, 138.47, 136.71, 131.12, 124.51,
120.06, 113.05, 107.81, 92.23, 60.13, 43.34, 31.50, 30.02, 23.40, 16.73,
14.35. MS (ESI) calcd for C20H24N4O4, m/z 384.1798, found
385.1950 (M + H)+.
for 20 were included in the structural model as idealized atoms
(assuming sp2- or sp3-hybridization of the carbon or nitrogen atoms
and C−H bond lengths of 0.95−1.00 Å or N−H bond lengths of 0.88
Å). The isotropic thermal parameters of all idealized hydrogen atoms
for 20 were fixed at values 1.2 (nonmethyl) or 1.5 (methyl) times the
equivalent isotropic thermal parameter of the carbon, nitrogen, or
oxygen atom to which they are covalently bonded.
Human TLR-3/-4/-5/-7/-8/-9 Reporter Gene Assays (NF-κB
Induction). The induction of NF-κB was quantified using human
TLR-3/-4/-5/-7/-8/-9-specific HEK-Blue reporter gene assays as
previously described by us.27,40,49 HEK293 cells stably cotransfected
with the appropriate hTLR and secreted alkaline phosphatase (sAP)
were maintained in HEK-Blue Selection medium containing zeocin
and normocin. Stable expression of secreted alkaline phosphatase
(sAP) under control of NF-κB/AP-1 promoters is inducible by
appropriate TLR agonists, and extracellular sAP in the supernatant is
proportional to 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 confluency was
achieved, and they were subsequently stimulated with graded
concentrations of stimuli. sAP was assayed spectrophotometrically
using an alkaline phosphatase-specific chromogen (present in HEK-
detection medium as supplied by the vendor) at 620 nm.
Synthesis of Compound (39): 2,2′-(Hexane-1,6-diylbis-
(azanediyl))bis(7-methyl-3-(pyridin-2-ylamino)furo[2,3-c]pyridine-
4,2-diyl)dimethanol. To a solution of 2-aminopyridine (48 mg, 0.50
mmol) in anhydrous acetonitrile were added pyridoxalhydrochloride
(112 mg, 0.56 mmol), 4 N HCl/dioxane (10 μL), and 1,6-
diisocyanohexane (36 μL, 0.24 mmol). The reaction mixture was
then heated under microwave conditions (600 W, 80 °C) in a sealed
vial for 2 min. After the reaction mixture was cooled to room
temperature, solvent was removed and the residue was purified using
Immunoassays for Cytokines. Fresh human peripheral blood
mononuclear cells (hPBMC) were isolated from human blood
obtained by venipuncture with informed consent and as per
institutional guidelines on Ficoll-Hypaque gradients as described
elsewhere.55 Aliquots of PBMCs (105 cells in 100 μL/well) were
stimulated for 12 h with graded concentrations of test compounds.
Supernatants were isolated by centrifugation and were assayed in
triplicate using analyte-specific multiplexed cytokine/chemokine bead
array assays as reported by us previously.56
1
column chromatography to obtain compound 39 (21 mg, 7%). H
Transcriptomal Profiling in Human PBMCs. Detailed procedures
for transcriptomal profiling have been described by us previously.49
Briefly, fresh human PBMC samples were stimulated with 10 μg/mL
of 37b and 37f for 2 h, and total RNA was extracted from 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 on 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 positive
controls, and a 5′/3′ ratio of <3.0. To facilitate direct comparison of
gene expression data between different samples, the GeneChip data
were first subjected to preprocessing. This step involved scaling (in
GCOS) 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 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.
Rabbit Immunization and Antigen-Specific ELISA. All experiments
were performed at Harlan Laboratories (Indianapolis, IN) in
accordance with institutional guidelines (University of Kansas
IACUC permit # 119-06). Cohorts of adult female New Zealand
White rabbits (n = 4 per cohort) were immunized intramuscularly in
the flank region with either 100 μg of bovine α-lactalbumin in 0.2 mL
of saline (unadjuvanted control) or 100 μg of bovine α-lactalbumin
plus 100 μg of 37b, 40, or 41 in 0.2 mL of saline. Preimmune test-
bleeds were first obtained via venipuncture of the marginal vein of the
ear. Animals were immunized on days 1, 15, and 28. A final test-bleed
was performed via the marginal vein of the ear on day 38. Sera were
stored at −80 °C until used. Bovine α-lactalbumin-specific ELISAs
were performed in 384-well format using automated liquid handling
methods as described by us.42
NMR (500 MHz, MeOD) δ 8.07 (t, J = 8.0 Hz, 2H), 7.97 (s, 2H),
7.90 (d, J = 5.6 Hz, 2H), 7.31 (d, J = 5.6 Hz, 2H), 7.07 (t, J = 6.7 Hz,
2H), 4.64 (s, 4H), 3.57 (t, J = 7.0 Hz, 4H), 2.75 (s, 6H), 1.78−1.65
(m, 4H), 1.46 (s, 4H). 13C NMR (126 MHz, MeOD) δ 165.67,
155.95, 145.66, 143.71, 142.10, 137.48, 133.70, 132.91, 124.87, 115.83,
115.43, 90.05, 59.50, 43.86, 30.65, 27.48, 13.02. MS (ESI) calcd for
C34H38N8O4, m/z 622.3016, found 623.3187 (M + H)+.
X-ray Crystallographic Structural Studies of 20, 28, and 37e.
Crystals for compound 28 utilize the noncentrosymmetric triclinic
space group P1−C1 with eight crystallographically independent
molecules in the asymmetric unit. They also invariably form multiply
twinned bundles. After many unsuccessful attempts, a multidomain
specimen of 28 was cut from one of these bundles that gave a set of
diffracted intensities that permitted a crystal structure solution but not
a satisfactory refinement. Full sets of unique diffracted intensities were
measured for single-domain specimens of compounds 20 and 37e
using monochromated Cu Kα radiation (λ = 1.54178 Å) on a Bruker
Proteum Single Crystal Diffraction System equipped with Helios
multilayer optics, an APEX II CCD detector, and a Bruker MicroStar
microfocus rotating anode X-ray source operating at 45 kV and 60 mA.
Diffracted intensities were obtained with the Bruker program SAINT,
and the structures were solved using “direct methods” techniques
incorporated into the Bruker SHELXTL Version 2010.3−0 software
package. All stages of weighted full-matrix least-squares refinement
were performed using the SHELXTL software with Fo2 data. The final
structural model for compounds 20 and 37e incorporated anisotropic
thermal parameters for all non-hydrogen atoms and isotropic thermal
parameters for all hydrogen atoms. The respective asymmetric units
consist of four molecules of 20 and one molecule of 37e. All hydrogen
atoms for 37e and hydrogen atoms of 20 that are bonded to amine
nitrogens (one in each molecule) were located in a difference Fourier
map and included in the structural model as independent isotropic
atoms whose parameters were allowed to vary in least-squares
refinement cycles. All hydroxyl hydrogens for 20 were placed at
idealized sp3-hybridized positions with an O−H bond length of 0.84 Å;
the hydroxyl group was then allowed to rotate about its O−C bond
during least-squares refinement cycles. The remaining hydrogen atoms
M
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(10) Shaw, M. H.; Reimer, T.; Kim, Y. G.; Nunez, G. NOD-like
receptors (NLRs): bona fide intracellular microbial sensors. Curr.
Opin. Immunol. 2008, 20, 377−382.
ASSOCIATED CONTENT
■
S
* Supporting Information
Characterization data (1H, 13C, mass spectra), LC-MS analyses
of final compounds, and summary crystallographic data of
compounds 20 and 37e. This material is available free of charge
via the Internet at http://pubs.acs.org.
(11) Kawai, T.; Akira, S. The role of pattern-recognition receptors in
innate immunity: update on Toll-like receptors. Nat. Immunol. 2010,
11, 373−384.
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innate receptors. J. Infect. Chemother. 2008, 14, 86−92.
(13) Takeda, K.; Akira, S. Toll-like receptors. Curr. Protoc. Immunol.
2007, Chapter 14, Unit.
Accession Codes
Crystal structures for compounds 20 and 37e have been
deposited in the Cambridge Crystallographic Data Centre
(CCDC). The deposition numbers are 893565 and 893566.
(14) Akira, S. Toll-like receptors and innate immunity. Adv. Immunol.
1902, 78, 1−56.
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proteins linking innate and acquired immunity. Nature Immunol. 2001,
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AUTHOR INFORMATION
Corresponding Author
*Tel: 785-864-1610. Fax: 785-864-1961. E-mail: sdavid@ku.
■
(16) Cottalorda, A.; Verschelde, C.; Marcais, A.; Tomkowiak, M.;
Musette, P.; Uematsu, S.; Akira, S.; Marvel, J.; Bonnefoy-Berard, N.
TLR2 engagement on CD8 T cells lowers the threshold for optimal
antigen-induced T cell activation. Eur. J. Immunol. 2006, 36, 1684−
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edu.
Notes
The authors declare no competing financial interest.
(17) Kaisho, T.; Akira, S. Toll-like receptors as adjuvant receptors.
Biochim. Biophys. Acta 2002, 1589, 1−13.
ACKNOWLEDGMENTS
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(18) Wu, W.; Li, R.; Malladi, S. S.; Warshakoon, H. J.; Kimbrell, M.
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R.; Warshakoon, H. J.; Malladi, S. S.; David, S. A. Structure-activity
relationships in toll-like receptor 2-agonists leading to simplified
monoacyl lipopeptides. J. Med. Chem. 2011, 54, 8148−8160.
(20) Salunke, D. B.; Shukla, N. M.; Yoo, E.; Crall, B. M.; Balakrishna,
R.; Malladi, S. S.; David, S. A. Structure-Activity Relationships in
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Chem. 2012, 55, 3353−3363.
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M.; Davies, D. R. Structural basis of toll-like receptor 3 signaling with
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U. F.; Seydel, U.; Zhringer, U.; Galanos, C.; Lderitz, O.; Westphal, O.;
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M. W.; Jeffery, E. W.; Johnson, D. A.; Lacy, M. J.; Baldridge, J. R.;
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Birmachu, W.; Bomersine, S. N.; Gibson, S. J.; Imbertson, L. M.;
Jacobson, J. R.; Knafla, R. T.; Maye, P. V.; Nikolaides, N.; Oneyemi, F.
Y.; Parkhurst, G. J.; Pecore, S. E.; Reiter, M. J.; Scribner, L. S.;
Testerman, T. L.; Thompson, N. J.; Wagner, T. L.; Weeks, C. E.;
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4465.
This work was supported by NIH/NIAID Contract
HSN272200900033C. We gratefully acknowledge NSF-MRI
Grant CHE-0923449, which supported the purchase of a
Bruker MicroStar Cu rotating anode diffractometer at the
University of Kansas Small-Molecule X-ray Crystallography
Laboratory.
ABBREVIATIONS USED
■
dsRNA, double-stranded RNA; EC50, half-maximal effective
concentration; ESI-TOF, electrospray ionization-time-of-flight;
HEK, human embryonic kidney; IL, interleukin; LPS, lip-
opolysaccharides; MHC, major histocompatibility complex;
MyD88, myeloid differentiation primary response gene 88; NF-
κB, nuclear factor-κB; PAMPs, pathogen associated molecular
patterns; PBMCs, peripheral blood mononuclear cells; PRRs,
pattern recognition receptors; SAP, secreted alkaline phospha-
tase; SAR, structure activity relationship; ssRNA, single-
stranded RNA; TLR, toll-like receptor; TNF-α, tumor necrosis
factor-α
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