Identification of 2,6-Disubstituted 3 H-Imidazo[4,5- b]pyridines as Therapeutic Agents for Dysferlinopathies through Phenotypic Screening on Patient-Derived Induced Pluripotent Stem Cells

Article abstract of DOI:10.1021/acs.jmedchem.9b01100

Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubst

Full text of DOI:10.1021/acs.jmedchem.9b01100

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Article
Identification of 2,6-Disubstituted-3#-imidazo[4,5-b]pyridines
as Therapeutic Agents for Dysferlinopathies through
Phenotypic Screening on Patient-Derived iPSCs
Hiroyuki Takada, Akira Kaieda, Michiko Tawada, Tomoko Nagino, Katsunori Sasa,
Tatsuo Oikawa, Akiko Oki, Tomoya Sameshima, Kazumasa Miyamoto, Makoto
Miyamoto, Yuko Kokubu, Ryuichi Tozawa, Hidetoshi Sakurai, and Bunnai Saito
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01100 • Publication Date (Web): 24 Sep 2019
Downloaded from pubs.acs.org on September 25, 2019
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Identification of
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2,6-Disubstituted-3H-imidazo[4,5-b]pyridines as
Therapeutic Agents for Dysferlinopathies through
Phenotypic Screening on Patient-Derived iPSCs
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Hiroyuki Takada,*,† Akira Kaieda,† Michiko Tawada,† Tomoko Nagino,‡ Katsunori Sasa,‡ Tatsuo
Oikawa,‡ Akiko Oki,† Tomoya Sameshima,† Kazumasa Miyamoto,† Makoto Miyamoto,† Yuko
Kokubu,§ Ryuichi Tozawa,‡ Hidetoshi Sakurai,§ and Bunnai Saito†
†Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome,
Fujisawa, Kanagawa 251-8555, Japan
‡T-CiRA Discovery, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi
2-chome, Fujisawa, Kanagawa 251-8555, Japan
§Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin,
Sakyo-ku, Kyoto 606-8507, Japan.
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ABSTRACT:
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Dysferlinopathies, which are muscular diseases caused by mutations in dysferlin gene, remain
serious medical problems due to the lack of therapeutic agents. Herein, we report the design,
synthesis, and structure-activity relationships of a 2,6-disubstituted-3H-imidazo[4,5-b]pyridine
series, which was identified from the phenotypic screening of chemicals that increase the level of
dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs).
Optimization studies with cell-based phenotypic assay led to the identification of a highly potent
compound 19 with dysferlin elevation effects at double-digit nanomolar concentrations. In
addition, the molecular target of our chemical series was identified as tubulin, through a tubulin
polymerization assay and a competitive binding assay using a photoaffinity labeling probe.
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Me
Me
N
N
O
S
N
H
N
H
O
N
N
1
19
EC150 = 5.9 M
EC150 = 0.012 M
Key words: small molecule, phenotypic screening, induced pluripotent stem cells (iPSCs),
dysferlinopathy, dysferlin (DYSF), tubulin, photoaffinity labeling (PAL) probe, target identification
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INTRODUCTION
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Cell-based phenotypic screening is an attractive drug discovery approach because it does not
require an established biological knowledge of molecular targets, and it leads to the discovery of
novel druggable targets and therapeutic agents.1, 2 In particular, phenotypic screening using
patient-derived induced pluripotent stem cells (iPSCs) is expected to be one of the most
disease-relevant evaluation systems.3-6 Dysferlinopathies are autosomal recessive muscular
disorders that are caused by mutations in the dysferlin gene.7, 8 Dysferlin is dominantly expressed
in skeletal or cardiac muscles and plays an essential role in the repair of damaged and disrupted
plasma membranes.9-11 The loss or reduction of dysferlin results in the weakening of muscles as
the muscle membrane is not restored sufficiently. Dysferlinopathies are clinically categorized
into at least three phenotypes based on the initial pattern of muscle weakness: limb girdle
muscular dystrophy 2B (LGMD2B), Miyoshi myopathy (MM), and distal myopathy with anterior
tibial onset (DMAT). Currently, there is no effective treatment for dysferlinopathies; therefore,
the development of novel therapeutic agents is highly desired.
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Many types of dysferlin mutations have been reported, and it is evident that dysferlin level in
human fibroblasts with the c.2997G>T (p.W999C) missense mutation is much lower than that in
normal human fibroblasts.12 Additionally, treatment with a proteasomal inhibitor, MG-132,
increased the level of p.W999C mutated dysferlin protein, and the membrane blebbing level was
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also restored depending on the increase in the level of mutated dysferlin. Therefore, it is
suggested that p.W999C mutated dysferlin protein possesses membrane resealing function.
Indeed, a clinical study reported that patients with the homozygous c.2997G>T mutation had late
onset of dysferlinopathies relative to those with the heterozygous c.2997G>T mutation.13 These
observations indicate that an increase in the level of p.W999C mutated dysferlin protein could
alleviate the symptoms of dysferlinopathies. Moreover, we previously developed a rapid,
efficient, and reproducible method to differentiate iPSCs into mature myocytes, and we
demonstrated its application in a disease model of MM.14 This method enabled us to establish an
assay system to screen chemicals that could increase the dysferlin level in myocytes
differentiated from MM patient-derived iPSCs.15 The pilot screening of 622 commercially
available compounds identified nocodazole, a tubulin inhibitor, as a hit compound, which was
found to restore membrane resealing function.
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Following the pilot screening, we carried out a more extensive high-throughput screening of
small molecules to find more potent and drug-like compounds. From a screen of structurally
diverse and drug-like small molecules (ca. 6400), we identified
2,6-disubstituted-3H-imidazo[4,5-b]pyridine 1 as a hit compound, which showed dysferlin
elevation effects in a dose-dependent manner. Compound 1 and its derivatives were originally
derived in Takeda’s legacy project, where these compounds exhibited the growth inhibition of
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HER2-positive cell lines such as SK-BR-3 and BT-474; however, their molecular target remains
unknown.16 In the present study, we describe the structure-activity relationships (SAR) of
2,6-disubstituted-3H-imidazo[4,5-b]pyridines and our efforts on the target deconvolution of our
chemical series.
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RESULTS AND DISCUSSION
In the dysferlin elevation assay,15 the threshold value for detecting an increase in the dysferlin
level was set as median + 3 standard deviations of vehicle controls, which converged to slightly
less than 1.5-fold relative to the median value of vehicle controls. Therefore, we defined the
indicator of potency, EC150, as a compound concentration at which the dysferlin level is increased
by 1.5-fold. As a result of the high throughput screening, we identified a hit compound 1, which
exhibited an EC150 value of 5.9 μM and acceptable physicochemical properties such as solubility
and membrane permeability. As our assay was a cell-based phenotypic screening, it is noteworthy
that compound 1 did not show any cytotoxic effect on HepG2 cells even at 100 μM. Thus, we
executed SAR studies in order to understand which features are crucial for cellular activity and
identify highly potent compounds. The SAR information is also required for target identification
approaches such as chemical proteomics or correlation analysis with target-based assay results17,
18; for example, it is crucial for the preparation of chemical probes to clarify which part of the
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compound turns toward the solvent region to attach a linker motif without disrupting the potency.
In developing the cell-based SAR using the dysferlin elevation assay, the structure of compound
1 was divided into three structural units: 1) C-2 substituents on imidazopyridine; 2) C-6 aryl
groups on imidazopyridine; 3) imidazopyridine core scaffold. We carried out systematic
modifications of each structural unit and evaluated the effects on the dysferlin level.
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1
Compound
C6
N
O
C2
Cellular activity: EC150 = 5.9 µM
Permeability: 332 nm/sec
Solubility: 6.4 µg/mL
N
H
N
Cytotoxicity: IC50 >100 µM
Figure 1. Structure of hit compound 1.
First, the effects of C-2 substituents on cellular activity were investigated. Replacing the
phenethyl group at C-2 with a phenyl (2) or benzyl (3) group resulted in the loss of cellular
activity. To confirm the tolerability of substitution on the terminal phenyl ring, a chlorine atom
was introduced into the ortho-, meta- and para-position, respectively. Among these,
para-substituted 6 showed a potency comparable to 1, whereas ortho- (4) or meta- (5) substituted
compounds showed reduced potency. The para-substitution was tolerated, and hence, a variety of
para-substitution was explored. Consequently, a fluorophenethyl group (9) increased potency by
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approximately 6-fold compared to hit compound 1. In contrast, an electron donating methyl (7) or
methoxy group (8) and an electron withdrawing trifluoromethyl (10) or cyano group (11) resulted
in a loss in potency, suggesting the size limitations of the binding pocket around the terminal
phenyl ring.
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With the potent compound 9 in hand, we explored the aryl group at C-6. While replacing the
furan ring with a benzene ring (12) hardly affected cellular activity, compound 13 containing a
thiophene ring exhibited an approximately 7-fold increase in potency as compared to 9. The
addition of a methyl group into the 3-position of the thiophene ring (14) was tolerated; however,
the substitution at the 4 or 5-position (15 or 16) resulted in a reduction of cellular activity. As in
compound 17, the ether-linked C-2 group resulted in an approximately 2-fold increase in potency
compared to the corresponding carbon-linked analogue 13. Surprisingly, the elimination of the
fluorine atom on the phenyl ring of compound 17 slightly enhanced the potency as in compound
18. In addition, it should be noted that compound 18 exhibited no cytotoxic effect as with the
case of hit compound 1. Given that the 3-position methyl group on the thiophene ring was
tolerated as in 14, a bulkier isopropyl group was introduced into the thiophene ring of compound
18, which led to an approximately 3-fold increase in potency (19).
Table 1. Structure-Activity Relationships of C-2 and C-6 Groups
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Ar
N
R
N
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H
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EC150 (M)a
Compound
R
Ar
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1
5.9 (3.3–10)
O
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>10
>10
O
O
Cl
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5
>10
>10
O
O
O
O
O
O
O
Cl
6
4.3 (2.4–7.6)
>10
Cl
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Me
MeO
F
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>10
9
1.0 (0.45–2.3)
>10
10
F3C
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>10
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0.15 (0.064–0.33)
0.11 (0.080–0.25)
1.9 (0.75–5.0)
S
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6.3 (1.5–26)
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0.067 (0.037–0.12)
0.039 (0.017–0.087)
0.012 (0.0058–0.023)
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a n = 8, 95 % confidence intervals shown in parentheses.
Next, we investigated the role of the imidazopyridine core in cellular activity.
Oxazolo[4,5-b]pyridines 20 and 21 exhibited a considerable loss in potency, indicating that the
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NH group of the imidazole ring might contribute to cellular activity by the formation of a
hydrogen bond with the target protein. Similarly, upon the removal of the nitrogen atom of the
pyridine ring, the resultant benzimidazole 22 did not exhibit a dysferlin elevation effect even at
10 μM. Furthermore, the introduction of a methyl group adjacent to the pyridine nitrogen resulted
in the loss of cellular activity as in 23. These results implied that the pyridine nitrogen possibly
made a hydrogen bond with the target protein, thereby contributing to cellular activity. In
contrast, the introduction of a methyl group at the 7-position of imidazopyridine ring was
relatively tolerated, as in 24. As a result, 3H-imidazo[4,5-b]pyridine was found to be the optimal
core structure for cellular activity.
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Table 2. Structure-Activity Relationships of the Center Ring
S
Het
F
EC150 (M)a
Compound
Het
N
13
0.15 (0.064–0.33)
N
H
N
N
O
20
21
>10
>10
N
N
O
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>10
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0.37 (0.17–0.82)
N
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N
H
a n = 8, 95 % confidence intervals shown in parentheses.
Furthermore, we sought to elucidate the plausible target of the chemical series. We previously
reported that the tubulin inhibitors, nocodazole and colchicine19, increased the level of dysferlin
in our dysferlin elevation assay.15 Accordingly, we proposed that the cellular activity of the
2,6-disubstituted-3H-imidazo[4,5-b]pyridine series would be mediated by the inhibition of
tubulin polymerization. The inhibitory effects of several analogues on tubulin polymerization
were evaluated in HepG2 cells to determine if they are correlated with dysferlin elevation effects
on iPSC-derived myocytes (for the structure of measured compounds, see Table S1, Supporting
Information). As hypothesized, the correlation plot suggests that the inhibition of tubulin
polymerization caused the elevation of dysferlin in the chemical series (Figure 2). It is worth
noting that the compound series did not exhibit inhibitory activities against kinases through
competitive binding assays, although 3H-imidazo[4,5-b]pyridine is a typical hinge binder.
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(Figure S1, Supporting Information)
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R² = 0.86
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Tubulin depolymerization pMEC
Figure 2. Correlation plot of pEC150 with tubulin depolymerization pMEC. MEC, minimal effective
concentration; R, correlation coefficient. The EC150 value of compounds described as square or triangle is
>10 μM. The MEC value of compounds described as circle or square is <0.080 μM or >50 μM,
respectively.
Encouraged by these results, we performed docking studies in order to further elucidate the
binding modes of the chemical series with tubulin molecules. It is reported that nocodazole binds
to the interface of alpha/beta-tubulin hetero dimers, which is known as the colchicine binding
site.20 Nocodazole contains an imidazole ring, which is an essential moiety for cellular activity in
our chemical series, and hence, we thought nocodazole and our chemical series might bind to the
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common binding site (Figure 3a). We conducted the docking calculation for compound 19 based
on the X-ray co-crystal structure of nocodazole with tubulin (PDB code: 5CA1) (Figure 3b-c).
Our docking analysis suggested that the NH-group of the imidazole ring made a hydrogen bond
with the back bone oxygen atom of Val236 of beta-tubulin. The loss in potency of
oxazolo[4,5-b]pyridines 20 and 21 should be attributed to the absence of this interaction. In
addition, the nitrogen atom of the pyridine ring formed a water-mediated hydrogen bond with
Gly235 and Cys239 of beta-tubulin. In the X-ray co-crystal structure of nocodazole, the water
molecule tightly binds to Gly235 and Cys239, and the water-mediated interaction is formed by
the carbonyl group on the thiophene ring (Figure 3d).20 These results are consistent with the
observation that benzimidazole 22 without the nitrogen atom had a considerable loss in potency.
The isopropyl group on the thiophene ring occupies the hydrophobic region consisting of
Leu246, Leu253, Asn256, and Met257 of beta-tubulin. This hydrophobic interaction is
considered to contribute to the high potency of compound 19. Generally, our docking model
explains the SAR of the chemical series.
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(a)
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N
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HN
O
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19
Compound
EC150 = 0.012 µM
Nocodazole
EC150 = 1.0 µM
(b)
(c)
(d)
(e)
Figure 3. (a) Structure and cellular activity of nocodazole and compound 19. (b) Docking model of 19
with an alpha/beta-tubulin hetero dimer. Compound 19 is light blue, alpha-tubulin is black, and
beta-tubulin is grey. (c) The binding site of 19. Residues that form hydrogen bonds or hydrophobic
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interactions with 19 are shown as sticks and are labeled. (d) X-ray co-crystal of nocodazole with tubulin
(PDB code: 5CA1). Residues that form hydrogen bonds with the ligand are shown as sticks and are
labeled. (e) Surface of residues within 4.0 Å of 19.
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Our docking model was found to be consistent with SAR, and hence, we tried to confirm the
direct binding between our compounds and tubulins using a photoaffinity labeling (PAL) probe,
which covalently binds to its target protein after ultraviolet irradiation.21 Our docking model
indicated that there was a long and narrow cavity exposed to the solvent region around the 3- and
4-positions of the thiophene ring, suggesting that a chemical probe linker could be attached
without disrupting the binding affinity to tubulins (Figure 3e). Thus, we introduced a linker motif
into the thiophene ring of compound 18 and evaluated its effect on the level of dysferlin. First, an
amide group was introduced because it is synthetically feasible to extend various linker parts by
amide coupling. Unlike in the case of the methyl or isopropyl group, a methylamide group at the
3-position of the thiophene ring resulted in a considerable loss of cellular activity (25). In
contrast, compound 26 containing a methylamide group at the 4-position sustained high potency.
Furthermore, a 2-(2-methoxyethoxy)ethylamide group was also tolerated, as in compound 27,
suggesting that the terminal alkoxy chain should turn toward the solvent region. Based on these
results, we synthesized a PAL probe 28, containing a photo-reactive diazirine group and an
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alkyne tag, which exhibited cellular activity at submicromolar concentrations.
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Table 3. Preparation of PAL probe 28
9
R
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
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54
55
56
57
58
59
60
N
H
O
N
EC150 (M)a
0.039 (0.017–0.087)
>10
Compound
R
18
25
S
NH
O
S
O
S
26
27
0.11 (0.038–0.31)
0.40 (0.096–1.7)
NH
O
O
O
S
NH
N
28
0.37 (0.20–0.69)
N
O
NH
S
a n = 8, 95 % confidence intervals shown in parentheses.
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With probe 28 and purified tubulins, a competitive binding assay was carried out. Commercially
available porcine brain tubulins were incubated with 28 after pre-incubation with or without
competitive compounds, followed by ultraviolet irradiation for crosslinking between 28 and
tubulins. Subsequently, the crosslinked tubulins were labeled with a fluorescent dye,
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tetramethylrhodamine (TAMRA), by alkyne-azide click-reaction. The tubulins were separated by
SDS-PAGE, and the images for fluorescently labeled tubulins and total tubulins were captured
(Figure 4). The fluorescently labeled tubulins were clearly observed without competitive
compounds, suggesting that probe 28 was effectively bound to the tubulins. In addition, the same
fluorescent band was not detected in the case where the porcine brain tubulins were pre-incubated
with compound 18 (EC150 = 0.039 μM) before the treatment with 28, whereas pre-incubation with
compound 21 (EC150 > 10 μM) hardly affected the fluorescent labeling. Furthermore, competitive
inhibition was observed when the porcine tubulins were pre-treated with colchicine binding site
inhibitors such as colchicine and nocodazole. In contrast, pre-incubation with vinblastine and
paclitaxel, which are known to bind to other biding sites19, did not inhibit the binding of probe
28. These results demonstrated that our chemical series increased the dysferlin level because of
tubulin depolymerization caused by direct binding to the colchicine binding site of tubulins.
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Figure 4. Image of SDS-PAGE for PAL probe-labeled tubulins. The tubulins were labeled with PAL
probe 28 after pre-incubation with non-labeled active or inactive compound (compound 18 or 21,
respectively) or known tubulin binders. PAL probe 28, which has an acetylene group, was probed with
TAMRA-azide by click reaction. (a) The samples were analyzed by SDS-PAGE, and the fluorescence
images were captured to detect the PAL-probe-labeled tubulins. (b) Subsequently, the gel was stained with
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Coomassie brilliant blue to visualize the total tubulin. (c) Structure and cellular activity of nocodazole and
vinblastine.
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CONCLUSIONS
In summary, we investigated the SAR of a 2,6-disubstituted-3H-imidazo[4,5-b]pyridine series
using a cell-based phenotypic screening in myocytes differentiated from MM patient-derived
iPSCs to identify compound 19, which exhibited dysferlin elevation effects at double-digit
nanomolar concentrations. The cellular activity of this chemical series correlated with their
inhibitory activity on tubulin polymerization. The competitive binding assay using purified
tubulins demonstrated that PAL probe 28 covalently bound to tubulins and it was competitively
inhibited by the active compound 18 or the colchicine binding site inhibitors, colchicine and
nocodazole. These observations indicated that 2,6-disubstituted-3H-imidazo[4,5-b]pyridines
increased the level of dysferlin through the inhibition of tubulin polymerization, mediated by
direct binding of the compounds to the colchicine binding site of tubulins. These results are
consistent with our previous work, where we demonstrated that nocodazole increased dysferlin
protein levels and improved the membrane resealing function. Considering that misfolded
proteins are transported along microtubules to form autophagosome in autophagy system, it is
suggested that the upregulation of dysferlin should be attributed to the blockage of the
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microtubule-dependent autophagy which might contribute to degradation of mutated dysferlin
protein. Indeed, upon the treatment of nocodazole, the accumulation of p62 and LC3 II were
observed, which indicated disruption of autophagy.15 Further investigation for the elucidation of
the detailed mechanism of action is underway. On the other hand, due to potential adverse effects
of microtubule disruption, such as cell cycle arrest, further optimization of the chemical series
was discontinued.22 However, we strongly believe that compound 19 can be used as a tool
compound to elucidate the detailed mechanism of action. Thus, it can contribute to the discovery
of druggable molecular targets and ultimately novel therapeutic agents for dysferlinopathies.
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CHEMISTRY
The synthesis of compound 17 is outlined in Scheme 1. Commercially available
5-bromo-2,3-diamino-pyridine was coupled with 2-(4-fluorophenoxy)acetic acid in the presence
of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
and N,N-diisopropylethylamine (DIPEA), followed by intramolecular condensation in the acidic
conditions to afford imidazopyridine intermediate 29. The intermediate 29 was converted to
compound 17 by Suzuki-Miyaura coupling with thiophen-2-ylboronic acid. Compounds 1-19 and
22-25 were prepared in a manner similar to Scheme 1.
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Scheme 1. Synthesis of compound 17a
6
7
8
9
a
b
H2N
H2N
Br
Br
N
N
S
N
H
N
H
F
O
F
O
N
N
N
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28
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17
29
aReagents and conditions: (a) (i) HATU, DIPEA, 2-(4-fluorophenoxy)acetic acid, DMF; (ii) AcOH,
reflux, 70 % (2 steps); (b) Pd(Amphos)Cl2, Na2CO3, thiophen-2-ylboronic acid, DME, water, microwave
120 °C, 17 %.
Compound 21 was prepared according to Scheme 2. Commercially available
3-(4-fluorophenyl)propanoic acid was converted to carboxamide 30, and the following
dehydration afforded the corresponding nitrile 31. Compound 31 was reacted with methanol
under acidic conditions to give orthoester 32, followed by condensation with
aminohydroxypridine 33 prepared from commercially available 2-amino-5-bromopyridin-3-ol via
Suzuki-Miyaura coupling to afford compound 21. Compound 20, a regioisomer of 21, was
prepared in a manner similar to Scheme 2.
Scheme 2. Synthesis of compound 21a
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9
O
O
OMe
OMe
OMe
a
d
b
c
N
F
OH
F
NH2
F
F
30
31
32
e
O
S
HO
Br
HO
S
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N
F
N
H2N
N
H2N
N
33
21
aReagents and conditions: (a) (i) (COCl)2, DMF (cat), CHCl3; (ii) NH3, CHCl3, 0 °C-RT, 76 % (2 steps);
(b) trifluoroacetic anhydride, 90 %; (c) (i) HCl gas, MeOH, Et2O, 0 °C-4 °C; (ii) MeOH, hexane; (d)
PdCl2(PPh3)2, Na2CO3, thiophen-2-ylboronic acid, 1,4-dioxane, water, 110 °C, 59 %; (e) AcOH, toluene,
reflux, 73 %.
The synthesis of PAL probe 28 is outlined in Scheme 3. The intermediate 34 was prepared
according to Scheme 1, and then hydrolyzed to give carboxylic acid 35. Compound 28 was
obtained through amide coupling between carboxylic acid 35 and
2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-amine. Compound 26 and 27 were prepared in a
manner similar to Scheme 3.
Scheme 3. Synthesis of compound 28a
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N
N
4
5
6
7
O
S
O
O
S
OMe
OH
NH
a
b
N
N
N
S
N
H
N
N
H
8
O
O
O
N
N
N
H
9
34
35
28
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18
19
20
21
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28
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aReagents and conditions: (a) NaOH aq., MeOH, THF, 86 %; (b) HATU, DIPEA,
2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-amine, DMF, 65 %.
EXPERIMENTAL SECTION
General Chemistry Information.
1H NMR spectra were recorded on Bruker AVANCE-300, 400, or 500 (300, 400, or 500 MHz).
Chemical shifts are given in parts per million (ppm) with tetramethylsilane as an internal
standard. Abbreviations used are as follows: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, dd = doublet of doublets, td = triplet of doublets, brs = broad singlet. Coupling
constants (J values) are given in hertz (Hz). The acidic protons were not frequently observed in
1H NMR spectra. Low-resolution mass spectra (MS) were acquired using an Agilent LC/MS
system (Agilent1200SL/Agilent6130MS) or Shimadzu UFLC/MS (Prominence UFLC high
pressure gradient system/LCMS-2020) operating in an electrosprayionization mode (ESI+). The
column used was an L-column 2 ODS (3.0 mm × 50 mm I.D., 3 μm, CERI) with a temperature of
40 °C and a flow rate of 1.2 or 1.5 mL/min. Condition 1: Mobile phases A and B under an acidic
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condition were 0.05% TFA in water and 0.05% TFA in MeCN, respectively. The ratio of mobile
phase B was increased linearly from 5 to 90% over 0.9 min, 90% over the next 1.1 min.
Condition 2: Mobile phases A and B under a neutral condition were a mixture of 5 mM AcONH4
and MeCN (9/1, v/v) and a mixture of 5 mM AcONH4 and MeCN (1/9, v/v), respectively. The
ratio of mobile phase B was increased linearly from 5 to 90% over 0.9 min, 90% over the next
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1
1.1 min. Chemical intermediates were characterized by H NMR and/or mass spectral data.
Unless otherwise stated, the purities of synthesized compounds for biological testing were >95%
determined by analytical HPLC. (The purities of 15, S4, and S6 were 94.9%, 91.9%, and 90.2%,
respectively.) Analytical HPLC were carried out using Corona CAD (Charged Aerosol Detector)
or photo diode array detector. The column was a Capcell Pak C18AQ (50 mm × 3.0 mm I.D.,
Shiseido, Japan) or L-column 2 ODS (30 mm × 2.0 mm I.D., CERI, Japan) with a temperature of
50 °C and a flow rate of 0.5 mL/min. Mobile phases A and B under a neutral condition were a
mixture of 50 mM AcONH4, water, and MeCN (1:8:1, v/v/v) and a mixture of 50 mM AcONH4
and MeCN (1:9, v/v), respectively. The ratio of mobile phase B was increased linearly from 5 to
95% over 3 min, 95% over the next 1 min. Mobile phases A and B under an acidic condition
were a mixture of 0.2% formic acid in 10 mM ammonium formate and 0.2% formic acid in
MeCN, respectively. The ratio of mobile phase B was increased linearly from 14 to 86% over 3
min, 86% over the next 1 min. Reaction progress was determined by thin layer chromatography
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(TLC) analysis on Merck Kieselgel 60 F254 plates or Fuji Silysia NH plates. Chromatographic
purification was carried out on silica gel columns (Merck Kieselgel 60, 70−230 mesh or 230−400
mesh, Merck; Chromatorex NH-DM 1020, 100−200 mesh, Fuji Silysia Chemical; Inject column
and Universal column, YAMAZEN, http://yamazenusa.com/products/columns/; or Purif-Pack Si
or NH, Shoko Scientific, http://shoko-sc.co.jp/english2/). Preparative HPLC was performed on a
Gilson Preparative HPLC System using a YMC-Actus Triart C18 column (150 mm × 20 mm
I.D., 5 μm, YMC). Condition 1: Mobile phases A and B under an acidic condition were 0.1%
TFA in water and 0.1% TFA in MeCN, respectively. Condition 2: Mobile phases A and B under
a neutral condition were 10 mM ammonium bicarbonate and MeCN, respectively. The ratio of
mobile phase B was increased linearly in 5-10 min. All commercially available solvents and
reagents were used without further purification. Yields were not optimized.
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6-(Furan-2-yl)-2-phenethyl-3H-imidazo[4,5-b]pyridine
6-bromo-2-phenethyl-3H-imidazo[4,5-b]pyridine (36)
(1).
A
mixture
0.497
of
(150
mg,
mmol),
2-(tributylstannyl)furan (0.18 M DMF solution, 5.0 mL), PdCl2(PPh3)2 (17 mg, 0.024 mmol) was
stirred at 80 °C under Ar for 18 h. The mixture was poured into water at room temperature and
extracted with EtOAc-THF. The organic layer was separated, washed with water and brine, dried
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over MgSO4, and concentrated in vacuo. The residue was purified by column chromatography
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8
1
(silica gel, eluted with 50% EtOAc in chloroform) to give 1 (72 mg, 50%) as a white solid. H
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NMR (300 MHz, DMSO-d6) δ 3.10-3.20 (4H, m), 6.62 (1H, td, J = 3.3, 1.8 Hz), 7.02 (1H, dd, J =
8.3, 3.3 Hz), 7.15-7.22 (1H, m), 7.24-7.30 (4H, m), 7.78 (1H, dd, J = 4.2, 1.2 Hz), 8.00-8.18 (1H,
m), 8.60-8.74 (1H, m), 12.56-13.01 (1H, m). MS m/z: 290.1 [M + H]+. HPLC purity 96.0%.
6-(Furan-2-yl)-2-phenyl-3H-imidazo[4,5-b]pyridine (2). Compound 2 was prepared from
6-bromo-2-phenyl-3H-imidazo[4,5-b]pyridine (37) in a manner similar to that described for the
synthesis of 1 and obtained (57% yield) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 6.65
(1H, dd, J = 3.4, 1.9 Hz), 7.07-7.10 (1H, m), 7.53-7.63 (3H, m), 7.81 (1H, dd, J = 1.8, 0.7 Hz),
8.19-8.28 (3H, m), 8.77 (1H, d, J = 2.0 Hz). MS m/z: 262.1 [M + H]+. HPLC purity 99.2%.
2-Benzyl-6-(furan-2-yl)-3H-imidazo[4,5-b]pyridine (3). Compound 3 was prepared from
2-benzyl-6-bromo-3H-imidazo[4,5-b]pyridine (38) in a manner similar to that described for the
synthesis of 1 and obtained (82% yield) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 4.21
(2H, s), 6.62 (1H, dd, J = 3.4, 1.8 Hz), 7.01 (1H, dd, J = 3.4, 0.7 Hz), 7.22-7.38 (5H, m), 7.78
(1H, dd, J = 1.8, 0.7 Hz), 8.11 (1H, brs), 8.68 (1H, d, J = 1.8 Hz), 12.70-13.04 (1H, m). MS m/z:
276.1 [M + H]+. HPLC purity 99.4%.
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2-(2-Chlorophenethyl)-6-(furan-2-yl)-3H-imidazo[4,5-b]pyridine (4).
A
mixture of
6
7
8
9
6-bromo-2-(2-chlorophenethyl)-3H-imidazo[4,5-b]pyridine (39) (200 mg, 0.594 mmol),
furan-2-ylboronic acid (100 mg, 0.894 mmol), Pd(PPh3)4 (60 mg, 0.052 mmol), 2 M Na2CO3 aq.
(1.8 mL) in toluene (4 mL), THF (1 mL) and DMF (2 mL) was stirred at 110 °C under Ar for 12
h. The mixture was poured into water at room temperature and extracted with EtOAc. The
organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography (silica gel, eluted with 33% -
100% EtOAc in hexane) to give 4 (90 mg, 47%) as a white solid. 1H NMR (300 MHz, DMSO-d6)
δ 3.13-3.29 (4H, m), 6.62 (1H, dd, J = 3.4, 1.8 Hz), 7.02 (1H, dd, J = 3.4, 0.7 Hz), 7.22-7.47 (4H,
m), 7.78 (1H, dd, J = 1.8, 0.7 Hz), 8.12 (1H, d, J = 1.9 Hz), 8.68 (1H, d, J = 2.0 Hz), 12.74-12.96
(1H, m). MS m/z: 324.1 [M + H]+. HPLC purity 98.7%.
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2-(3-Chlorophenethyl)-6-(furan-2-yl)-3H-imidazo[4,5-b]pyridine (5). Compound 5 was
prepared from 6-bromo-2-(3-chlorophenethyl)-3H-imidazo[4,5-b]pyridine (40) in a manner
similar to that described for the synthesis of 4 and obtained (17% yield) as a pale yellow solid. 1H
NMR (300 MHz, DMSO-d6) δ 3.17 (4H, s), 6.62 (1H, dd, J = 3.4, 1.8 Hz), 7.02 (1H, dd, J = 3.4,
0.7 Hz), 7.21-7.38 (4H, m), 7.78 (1H, dd, J = 1.8, 0.7 Hz), 8.11 (1H, d, J = 1.7 Hz), 8.67 (1H, d, J
= 2.0 Hz), 12.59-13.00 (1H, brs). MS m/z: 324.2 [M + H]+. HPLC purity 97.7%.
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2-(4-Chlorophenethyl)-6-(furan-2-yl)-3H-imidazo[4,5-b]pyridine (6). Compound 6 was
prepared from 6-bromo-2-(4-chlorophenethyl)-3H-imidazo[4,5-b]pyridine (41) in a manner
similar to that described for the synthesis of 4 and obtained (28% yield) as a pale yellow solid. 1H
NMR (300 MHz, DMSO-d6) δ 3.15 (4H, s), 6.62 (1H, dd, J = 3.4, 1.8 Hz), 7.02 (1H, d, J = 3.0
Hz), 7.26-7.36 (4H, m), 7.78 (1H, d, J = 1.2 Hz), 8.14 (1H, brs), 8.66 (1H, brs), 12.51-13.02 (1H,
m). MS m/z: 324.1 [M + H]+. HPLC purity 98.7%.
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6-(Furan-2-yl)-2-(4-methylphenethyl)-3H-imidazo[4,5-b]pyridine (7). Compound 7 was
prepared from 6-bromo-2-(4-methylphenethyl)-3H-imidazo[4,5-b]pyridine (42) in a manner
1
similar to that described for the synthesis of 4 and obtained (63% yield) as a white solid. H
NMR (300 MHz, DMSO-d6) δ 2.24 (3H, s), 3.11 (4H, brs), 6.58-6.66 (1H, m), 6.98-7.19 (5H, m),
7.78 (1H, s), 7.99-8.21 (1H, m), 8.59-8.76 (1H, m), 12.50-13.13 (1H, m). MS m/z: 304.3 [M +
H]+. HPLC purity 100.0%.
6-(Furan-2-yl)-2-(4-methoxyphenethyl)-3H-imidazo[4,5-b]pyridine (8). Compound 8 was
prepared from 6-bromo-2-(4-methoxyphenethyl)-3H-imidazo[4,5-b]pyridine (43) in a manner
1
similar to that described for the synthesis of 4 and obtained (28% yield) as a white solid. H
NMR (300 MHz, DMSO-d6) δ 3.02-3.16 (4H, m), 3.70 (3H, s), 6.62 (1H, dd, J = 3.4, 1.8 Hz),
6.80-6.87 (2H, m), 7.02 (1H, d, J = 4.1 Hz), 7.14-7.21 (2H, m), 7.78 (1H, dd, J = 1.8, 0.7 Hz),
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Journal of Medicinal Chemistry
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8.10 (1H, d, J = 1.8 Hz), 8.66 (1H, d, J = 2.0 Hz), 12.40-13.06 (1H, m). MS m/z: 320.2 [M + H]+.
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HPLC purity 99.2%.
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2-(4-Fluorophenethyl)-6-(furan-2-yl)-3H-imidazo[4,5-b]pyridine (9). Compound 9 was
prepared from 6-bromo-2-(4-fluorophenethyl)-3H-imidazo[4,5-b]pyridine (44) in a manner
1
similar to that described for the synthesis of 4 and obtained (51% yield) as a white solid. H
NMR (300 MHz, DMSO-d6) δ 3.14 (4H, s), 6.62 (1H, dd, J = 3.3, 1.8 Hz), 6.99-7.14 (3H, m),
7.25-7.34 (2H, m), 7.78 (1H, s), 7.99-8.20 (1H, m), 8.60-8.75 (1H, m), 12.52-13.01 (1H, m). MS
m/z: 308.1 [M + H]+. HPLC purity 96.9%.
6-(Furan-2-yl)-2-(4-(trifluoromethyl)phenethyl)-3H-imidazo[4,5-b]pyridine (10). Compound
10 was prepared from 6-bromo-2-(4-(trifluoromethyl)phenethyl)-3H-imidazo[4,5-b]pyridine (45)
in a manner similar to that described for the synthesis of 4 and obtained (48% yield) as a white
1
solid. H NMR (300 MHz, DMSO-d6) δ 3.19-3.26 (4H, m), 6.62 (1H, dd, J = 3.4, 1.8 Hz), 7.02
(1H, dd, J = 3.4, 0.7 Hz), 7.50 (2H, d, J = 7.9 Hz), 7.65 (2H, d, J = 8.0 Hz), 7.78 (1H, dd, J = 1.8,
0.7 Hz), 8.11 (1H, d, J = 2.0 Hz), 8.67 (1H, d, J = 2.0 Hz). MS m/z: 358.3 [M + H]+. HPLC
purity 99.0%.
4-(2-(6-(Furan-2-yl)-3H-imidazo[4,5-b]pyridin-2-yl)ethyl)benzonitrile (11). Compound 11
was prepared from 4-(2-(6-bromo-3H-imidazo[4,5-b]pyridin-2-yl)ethyl)benzonitrile (46) in a
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