Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.6b00619

The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma.

Full text of DOI:10.1021/acs.jmedchem.6b00619

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Article
Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-
imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally
Bioavailable, Selective, and Potent Allosteric AKT Inhibitor
Jean-Marc Lapierre, Sudharshan Eathiraj, David Vensel, Yanbin Liu, Cathy O Bull,
Susan Cornell-Kennon, Shin Iimura, Eugene W. Kelleher, Darin E. Kizer, Steffi
Koerner, Sapna Makhija, Akihisa Matsuda, Magdi Moussa, Nivedita Namdev, Ronald
E. Savage, Jeff Szwaya, Erika Volckova, Neil Westlund, Hui Wu, and Brian Schwartz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00619 • Publication Date (Web): 15 Jun 2016
Downloaded from http://pubs.acs.org on June 18, 2016
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Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-
yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric
AKT Inhibitor
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,†ǂ
†ǂ
†
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†
JeanꢀMarc Lapierre* , Sudharshan Eathiraj , David Vensel , Yanbin Liu , Cathy O. Bull ,
†
‡
†
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†
Susan CornellꢀKennon , Shin Iimura , Eugene W. Kelleher , Darin E. Kizer , Steffi Koerner ,
†
‡
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†
†
Sapna Makhija , Akihisa Matsuda , Magdi Moussa , Nivedita Namdev , Ronald E. Savage , Jeff
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Szwaya , Erika Volckova , Neil Westlund , Hui Wu , and Brian Schwartz
†
ArQule Inc., One Wall Street, Burlington, MA, 01803
‡
Daiichi Sankyo Co., Shinagawa R&D Center, 1ꢀ2ꢀ58, Hiromachi, Shinagawaꢀku, Tokyo 140ꢀ
8710, Japan
ǂ
Both coꢀauthors contributed equally to the manuscript
Abstract
The work in this paper describes the optimization of the 3ꢀ(3ꢀphenylꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ
2
ꢀyl)pyridinꢀ2ꢀamine chemical series as potent, selective allosteric inhibitors of AKT kinases,
leading to the discovery of ARQ 092 (21a). The coꢀcrystal structure of compound 21a bound to
fullꢀlength AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role
of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against
AKT1, AKT2 and AKT3, as well as potent cellular inhibition of AKT activation and the
phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent
inhibitor of the AKT1ꢀE17K mutant protein and inhibited tumor growth in a human xenograft
mouse model of endometrial adenocarcinoma.
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Introduction
The AGC kinases AKT1, AKT2 and AKT3 are key mediators of the PI3K/AKT/mTOR
signaling pathway, which promotes diverse physiological processes such as proliferation,
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migration, antiꢀapoptotic survival and protein synthesis. The activation of the PI3K/AKT/mTOR
signaling network is commonly observed in several human cancers and can be induced through a
variety of mechanisms, including the overexpression of upstream receptors, PI3KCAꢀactivating
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mutations, the loss of PTEN function, and the overexpression or mutational activation of AKT.
The serine/threonine AKT kinases represent a pivotal node connecting the PI3K and mTOR
pathways, suggesting that AKT kinases are compelling targets for the treatment of human
cancers and nonꢀoncology pathologies. Multiple efforts have been made to discover small
molecule inhibitors of AKT, including both allosteric inhibitors that target fullꢀlength AKT and
ATPꢀcompetitive inhibitors that are designed to bind the ATP binding pocket of the catalytic
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domain. Allosteric inhibitors offer a unique advantage over ATPꢀcompetitive inhibitors, in that
very high kinase selectivity can be achieved by targeting a distinct allosteric pocket in AKT
formed by the pleckstrin homology (PH) and kinase domains. In contrast to allosteric inhibitors,
which inhibit the phosphorylation of Thr308 and Ser473 in AKT, ATPꢀcompetitive inhibitors
paradoxically induce the hyperꢀphosphorylation of these two regulatory sites in cells and in vivo;
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however, the clinical significance of this observation is not yet understood.
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We recently reported the preꢀclinical characterization of ARQ 092 (21a) , a highly selective,
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allosteric inhibitor of AKT. Compound 21a showed strong affinity for unꢀphosphorylated fullꢀ
length AKT1 and potently inhibited the phosphorylated form of fullꢀlength AKT isoforms. In a
large panel of cell lines derived from various tumor types, 21a showed potent antiꢀproliferative
activity in cell lines containing PIK3CA/PIK3R1 mutations compared to those with wildꢀtype
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wt) PIK3CA/PIK3R1 or PTEN loss. Moreover, 21a also exhibited strong antiꢀtumor activity in
mouse xenograft models implanted with cell lines carrying a PIK3CA mutation and in a patientꢀ
derived tumor mouse model harboring an AKT1ꢀE17K activating mutation. Both animal studies
demonstrated the desirable pharmacokinetic properties of 21a which is under investigation in
clinical trials targeting advanced solid tumors and recurrent malignant lymphoma. Compound
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1a is also being investigated for treatment of Proteus syndrome, a disease driven by AKTꢀE17K
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mutation.
In our previous study, we demonstrated that optimizing activity against AKT1, AKT2 and
AKT3 was critical to achieving cellular potency; however, the most potent benzamide analogs of
the imidazo[4,5ꢀb]pyridine series had very low exposure in the plasma and tumors in mouse
xenograft models. Therefore, the final phase of the optimization was then focused on optimizing
the pharmacokinetics and demonstrating in vivo activity in tumor xenografts. One critical
optimization step in the early stage of discovery of this chemical series was the reduction to a
methyl group (4a) from a 4ꢀtꢀbutyl group observed in the initial hit (4b), followed by the
addition of a primary anime to the methyl group (6). These two small sideꢀchain modifications
significantly improved the biochemical potency of the compounds and incorporated the
substituents necessary for further optimization. Compound 6 became the cornerstone from which
an extensive optimization campaign was undertaken, part of which has been previously disclosed
with a focus on substitutions at the 5ꢀ and 6ꢀposition of the imidazo[4,5ꢀb]pyridine ring system,
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as well as the substitution of the benzylic amine amide.
In this communication, we describe the additional structureꢀactivity relationships (SARs) that
led to the discovery of 21a, its characterization in vitro and in vivo and its binding mode based
on the coꢀcrystallization of 21a with AKT1. To probe the structural features associated with this
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highly biologically active scaffold, we previously reported the screening and early optimization
of a series of 3ꢀ(3ꢀphenylꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀamines as potent AKT
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inhibitors. The SARꢀguided optimization of the imidazo[4,5ꢀb]pyridine series using in vitro and
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in vivo assessments yielded highly selective AKT inhibitors. In vivo characterization employed
both pharmacokinetic and pharmacodynamic analyses, and the lead inhibitors demonstrated oral
bioavailability in mice and were well tolerated, with robust inhibition of AKT activation, as well
as downstream AKT signaling. The screening paradigm in our previous study used a biophysical
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thermal shift assay to identify binders of AKT1 , followed by a biochemical assay using the unꢀ
phosphorylated AKT1 protein to recapitulate the binding event measured by the thermal shift
assay. This was achieved by preꢀincubating the test compounds with the unꢀphosphorylated
protein, followed by in situ activation. AKT activation entails the translocation of the protein to
the cell membrane and the binding of the PH domain of AKT to phosphoinositides, upon which a
conformational change permits the phosphorylation of Thr308 (for AKT1) in the activation loop
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by phosphoinositideꢀdependent kinase 1 (PDK1). The full activation of AKT is achieved when
Ser473 (for AKT1) in the hydrophobic motif (HM) domain is phosphorylated by the mTORC2
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complex. For the biochemical assay, unꢀphosphorylated fullꢀlength proteins (AKT1 (1ꢀ480),
AKT2 (1ꢀ481) and AKT3 (1ꢀ479)) were preꢀincubated with the test compounds, followed by the
successive addition of the activating proteins PDK1 and MAPKAPK2 (a Ser473ꢀphosphorylating
kinase that replaces the mTORC2 complex), lipid vesicles, and ATP before addition of the
substrate, biotinylated crosstide. The biochemical activities of the test compounds described in
this work against the two activating kinases (PDK1 and MAPKAPK2) were determined to
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ascertain whether the observed AKT inhibition was due specifically to AKT binding.
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Additionally, the biochemical inhibition of fullꢀlength, preꢀactivated AKT1, AKT2 and AKT3
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was probed for all compounds to further validate the inhibitory potency.
Synthesis
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From commercially available 2ꢀchloroꢀ3ꢀnitropyridine (1a) and substituted 2ꢀchloroꢀ3ꢀ
nitropyridines (1b-f), the displacement of the chlorine atom with substituted anilines in DMSO
or 1,4ꢀdioxane in the presence of a mild base (diisopropylethylamine or trimethylamine), under
heating, provided compounds 2a-c and 3a-e with good yields (Schemes 1 and 2). A oneꢀpot
condensation/cyclization of the substituted 2ꢀanilinoꢀ3ꢀnitropyridines (2a-c, 3a-e) with 2ꢀaminoꢀ
nicotinaldehyde under reducing conditions using sodium hydrosulfite in a DMSO/methanol
mixture (2:1) at 100 °C afforded compounds 4a-c and 5a-e with yields varying from 25ꢀ75%.
Intermediates 4c and 5a-e were Bocꢀdeprotected using 4 M HCl in 1,4ꢀdioxane at room
temperature, producing 6 and 7a-e in quantitative yields as HCl salts.
As shown in Scheme 3, intermediate 5d was reacted with phenyl boronic acid, 3ꢀ
acetamidophenyl boronic acid or 4ꢀacetamidophenyl boronic acid in a Suzuki coupling reaction,
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affording compounds 8a-d with good yields. Standard Bocꢀdeprotection with HCl produced
compounds 9a-d in quantitative yields as HCl salts. Alternatively, intermediate 5e was reacted
with (pyridinꢀ3ꢀyl)boronic acid under the same Suzuki conditions, affording the Bocꢀprotected
compound 8e with a substituent at the 6ꢀposition. Standard deprotection with HCl yielded 9e.
Synthesis of the gemꢀdimethyl compound 17 is depicted in Scheme 4. Racemic 2ꢀmethylꢀ2ꢀ
propanesulfinamide (10) was reacted with acetone in the presence of Ti(OEt) , affording the Nꢀ
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propanꢀ2ꢀylidene 11 with a 47% yield. Halogenꢀmetal exchange using 4ꢀbromoꢀN,Nꢀ
bis(trimethylsilyl)aniline and BuLi at a low temperature generated the 4ꢀlithio species, which
was trapped with 11. In situ cleavage of the SiꢀN bonds afforded the aniline 12 with a modest
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yield. Following a displacement reaction using 1e, the resulting compound 13 was subjected to a
deꢀprotection/reꢀprotection sequence, yielding the Bocꢀprotected intermediate 14 with excellent
yields. Cyclization of 14 with 2ꢀaminonicotinaldehyde under reducing conditions produced 15
with moderate yields. As described above, a Suzuki coupling reaction yielded 16, and a standard
Bocꢀdeprotection using HCl gave the final product 17.
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The synthesis of the aminoꢀcyclobutyl compounds 21a-c, 22 and 23 is shown in Scheme 5. The
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preparation of tertꢀbutyl (1ꢀ(4ꢀamino phenyl)cyclobutyl)carbamate is described and is now
commercially available. Using the starting nitropyridine 1e and tertꢀbutyl (1ꢀ(4ꢀamino
phenyl)cyclobutyl)carbamate in a displacement reaction produced 18 with moderate yields.
Cyclization under reducing conditions with 2ꢀaminonicotinaldehyde afforded the intermediate
19. A Suzuki coupling reaction using phenyl boronic acid, 3ꢀacetamidophenyl boronic acid or 4ꢀ
acetamidophenyl boronic acid produced 20a-c with good yields. Standard Bocꢀdeprotection with
HCl afforded the final compounds 21a-c in quantitative yields. Compound 22 was the result of a
conventional amine acylation using acetic anhydride in pyridine. Subjecting the deprotected
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compound 21b to HCl in dioxane for an extended period (3 days at 50 C) produced the deꢀ
acylated compound 23.
The replacement of the amino group appending the fourꢀmembered ring with a carboxylic acid
methyl ester (26) or a carboxamide (27) is shown in Scheme 6. Suzuki coupling (24→25) was
executed prior to the reductive cyclization with the 2ꢀaminoꢀnicotinaldehyde, and compound 26
was obtained with a good overall yield (46% over three steps). Hydrolysis followed by EDC
coupling produced 27 with a modest yield.
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Results and Discussion
The structureꢀactivityꢀrelationship described here was focused on two regions of the 3ꢀ(3ꢀ
phenylꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀamine series: the substitution around the
pyridine ring and substitution at the 4ꢀposition of the phenyl ring. The SAR of substitutions of
the pyridine ring is shown in Table 2. The introduction of a methyl group at the 7ꢀ (7a) or 6ꢀ
position (7b) led to improved potency; however, methyl groups at the 5ꢀ or 6ꢀposition (7c) were
not tolerated. A similar effect was noticed in previous SAR studies using the benzylamine
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amides , which showed little difference between the two positions on the imidazo[4,5ꢀb]pyridine
core. Compounds 7a and 7b displayed very modest inhibitory activity against AKT2 and AKT3.
The presence of a chlorine atom at the 5ꢀposition (7d) or a bromine atom at the 6ꢀposition (7e)
did not significantly affect the potency against AKT1. Interestingly, the introduction of a phenyl
ring at the 5ꢀposition dramatically improved the activity against all three isoforms, while a
pyridyl substitution at the 6ꢀposition also increased potency against AKT1 but spared AKT2 and
AKT3 by 100 fold and >300 fold, respectively.
After identifying the key position for improving activity against the AKT isoforms,
substitutions at the 5ꢀposition were further explored. A limited set of substitutions is exemplified
with compounds 9b, 9c and 9d. The introduction of more polar groups at the para or meta
position was well tolerated and offered similar levels of AKT inhibition and isozyme selectivity.
Combining the SAR points from compounds 4b, 6 and 9a, we were determined to make the
hybrid compound 17, which proved to be more arduous than anticipated. A small amount was
finally isolated, and the compound showed potent inhibitory activity against AKT1 (Table 3,
IC = 0.0054 ꢀM). Further investigation into substituting the benzylic position led to compound
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all three AKT isoforms remarkably improved. The role of the free amino group was confirmed
when we tested the acetylated compound 22, which showed a 10ꢀfold loss of potency against
AKT1. Replacing the acetamide group in 22 with a reverse acetamide (27) was tolerated and
showed similar potency; however, the introduction of an ester moiety (26) at this position was
deleterious. A similar SAR trend was observed in our previous study: the acetamide analogue of
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was active compared to nonꢀpolar or polar substituents that lacked hydrogen donors
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compound 8d of reference 11, IC = 0.81 ꢀM). Thus, the introduction of substituents at the
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benzylic position appeared to change the previously defined SAR when a basic nitrogen is
present.
The extension of the cyclobutylamine to compound 9b to produce 21b showed comparable
inhibitory activity to that of 21a against all AKT isozymes (Table 4). The removal of the
acetamide (23) did not change the IC50 values against inactive AKT. Finally, introducing a
pyridine nitrogen in the aromatic ring of the 5ꢀposition substituent (21c) modestly affected the
activities against AKT2 and AKT3.
The biochemical potencies of the compounds against inactive, unꢀphosphorylated AKT were
compared with those against active, phosphorylated AKT isoforms in the final stage of
optimization. As shown in Table 4, with the exception of 21c, the compounds showed similar
inhibitory potency for the enzymatically distinct forms of AKT. The presence of a nitrogen atom
in the aromatic substituent at position 5 appeared to be detrimental to the activity against AKT2
and AKT3. In contrast, as reported in our previous study, the initial hits of this chemical series
were preferentially highly active in the assay utilizing only the unꢀphosphorylated form of AKT.
Therefore, further lead optimization was continued with the same assay format. Finally, the fully
optimized, highly potent inhibitors were capable of inhibiting both the active and inactive forms
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of AKT with equivalent potency, and we believe that this functionality in the inhibitors is
necessary to target constitutive AKT kinase activity in cancer cells.
To define the binding mode of these potent AKT inhibitors and to understand the interactions of
the phenyl cyclobutylamine and phenyl groups at the 5ꢀposition, the crystal structure of unꢀ
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phosphorylated AKT1 was determined in complex with compound 21a (Figure 1). The overall
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conformation of AKT1 was very similar to the previously determined structure , adopting an
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identical autoꢀinhibitory conformation , and the inhibitor (21a) occupied an allosteric pocket at
the interface between the kinase and PH domains. The aminopyridine moiety of the (3ꢀphenylꢀ
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Hꢀimidazo[4,5ꢀb]ꢀpyridinꢀ2ꢀyl)pyridinꢀ2ꢀamine core forms hydrogen bonds with the disordered
αCꢀhelix and the β4ꢀstrand, and the core forms a key hydrophobic interaction with Trp80 in the
PH domain. The phenyl ring of phenylcyclobutylamine makes a direct hydrophobic contact with
Tyr272 in the highlyꢀconserved YRD motif of the kinase domain. The primary amine of
cyclobutylamine forms bidentate hydrogen bonds with the main chain carbonyl of Tyr 72 and the
carboxylate group of Asp274, and this interaction facilitates the steric orientation of the
cyclobutyl group towards the hydrophobic side chain of Ile 84 and the methyl group of Thr82 in
the PH domain. Therefore, a strong proton donor on the benzylic group is critical for the
interaction; thus, replacing this side chain with either with a single proton donor group, such as
an amide in 27, or a strong proton acceptor, such as the methyl ester group in 26, would be
detrimental. The phenyl moiety at the 5ꢀposition contacts the acyl side chains of Gln79 and
Asn53, and thus, polar substituents (9b, 9c, 9d, 21b and 23) on this hydrophobic phenyl ring are
well tolerated, as these groups project towards the solvent. The crystal structure further explains
the advantage of these interactions that led to activity improvement at the 5ꢀ position but not at
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the 6ꢀ or 7ꢀpositions, which is exemplified by 7a, 7b and 7c, where the methyl substituents are
directly exposed to the highly polar solvent region.
We next examined the in vitro antiꢀproliferative activity of the compounds in multiple cell lines
where the PI3K/AKT pathway is upregulated due to mutational activation; A2780 (ovarian
carcinoma), AN3CA (endometrial adenocarcinoma), IGROVꢀ1 (ovarian adenocarcinoma) and
LNCaP (prostate adenocarcinoma) were all monitored using a standard MTS assay. Compound
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9a exhibited micromolar antiꢀproliferative activity (Table 5), while its cyclobutylamine analog
showed a 10ꢀ35 fold increase in activity, being most potent against the IGROVꢀ1 cell line. The
acetamide analog 21b was less active against the A2780, AN3CA and LNCaP cell line yet was
best at inhibiting the proliferation of IGROVꢀ1 ovarian cells (IC50 = 0.16 ꢀM). The
corresponding amine 23 was also very potent in the MTS assay. Consistent with its biochemical
inhibition of AKT, the pyridine analog 21c was markedly less active in the cell lines, with the
exception of IGROVꢀ1 cells.
We assessed the ability of compounds 21a-c to inhibit AKT1 phosphorylation at residues S473
and T308, as well as the downstream PRAS40 residue T246 in cells using Western blot analysis.
Compounds 21a and 21b showed excellent inhibition of pꢀAKT (S473) and pꢀAKT (T308) in
both AN3CA and A2780 cells (Table 6). The inhibition of the downstream protein pꢀPRAS40
(T246) was observed with 21a (IC = 0.31 ꢀM), whereas 21b did not significantly inhibit the
5
0
phosphorylation of PRAS40 (IC = >3 ꢀM).
5
0
Table 7 shows CYP450 isoform inhibition and microsomal stability using human, mouse and
dog liver microsome preparations. Compounds 9a-b, 21a-c and 23 did not significantly inhibit
CYP450 1A2, 2C8, 2D6 and 3A4. Compounds 9a and 9b inhibited CYP450 2C9 with a subꢀ
micromolar IC value, while 9a also inhibited CYP450 2C19 (IC = <1 ꢀM). All compounds
5
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were adequately stable in liver microsomes (human, mouse and dog). In general, we found that
compounds from this chemical series possess adequate in vitro ADME properties, and compound
21a in particular showed good biochemical inhibition, cellular knockdown of AKT
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phosphorylation and ADME properties. In a mouse pharmacokinetic study, (po at 100 mg/kg, iv
at 5 mg/kg), compound 21a showed an oral bioavailability of 23%. An in vivo pharmacodynamic
assessment of 21a using NCrꢀM nude mice implanted with AN3CA tumor xenografts was
6
reported in our recent publication. Compound 21a resulted in 99%, 95% and 58% reductions in
pꢀAKT (S473), pꢀAKT (T306) and pꢀPRAS40 (T246), respectively, after tumorꢀbearing mice
were treated with 100 mg/kg po. The inhibition of phosphorylation was sustained at eight hours.
The plasma concentration of compound 21a at one hour was 2.1 ꢀM and decreased to 0.26 ꢀM
at 8 hours, while in the tumor, the concentration was 21.0 ꢀM at one hour and 9.6 ꢀM at 8 hours.
The concentrations in the tumor tissues were significantly higher than in the plasma, indicating a
marked preference for tissue accumulation compared with the vasculature compartment.
The efficacy of compound 21a has been probed in a previous communication, in which it was
shown to have strong tumor growth inhibition in mouse xenografts of endometrial, breast cancer
6
cell lines and patientꢀderived tumor models.
Definitive single dose pharmacokinetic studies were conducted in rats and monkeys (Table 9).
Compound 21a showed good absolute oral bioavailability in rats and monkeys with F values of
62% and 49%, respectively. The compound was more slowly absorbed in rats compared to
monkeys with Tmax values of 8.0 hr for rats versus 4.3 hr for monkeys. The halfꢀlife was also
longer in rats compared to monkeys with t1/2 values of 17 hr in rats versus 7 hr in monkeys. The
Cmax was 198 and 258 ng/mL and the AUCinf was 5496 and and 2960 hr*ng/mL in rats and
monkeys, respectively.
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In summary, compound 21a is a highly optimized drug candidate with desirable biochemical
potency and cellular inhibitory activity across multiple tumor types, as well as sustained in vivo
target inhibition and good oral bioavailability. Consistent with targeting a distinct allosteric site,
as revealed by its coꢀcrystal structure with AKT1, 21a demonstrated a very high selectivity when
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profiled against a large panel of kinases.
Experimental Section
Chemistry: All reagents and solvents were purchased from commercially available sources and
used without further purification. All reactions were carried out according to the indicated
procedures and conditions. Reactions were monitored by LC/MS analysis and/or thinꢀlayer
chromatography (TLC) on silicaꢀcoated glass plates (EMD silica gel 60 F ) with the indicated
254
eluent. The compounds were visualized using UV light (254 nm). LC/MS analysis was
performed on a Shimadzu HPLC/UV (214 nm and/or 254 nm wavelength) system coupled to
ELSD (Sedex 75, Sedere) and MS (ZQ, Micromass) detectors. Compounds were dissolved in
1
00% DMSO and separated on a Zorbax SBꢀC8 rapid resolution cartridge (30 × 4.6 mm, 3.5 µm,
mL/min flow rate) using acetonitrile/water as the mobile phase with 0.1% TFA as a modifier.
3
The gradient started at 5% acetonitrile and increased linearly to 95% acetonitrile over 1.9 min,
with a 0.3 min hold at 95%, and the cartridge was reꢀequilibrated to the original conditions in a
total of 2.5 min. The purity of the compounds selected for the in vivo studies was determined
using an Agilent HP1100 system with a YMC C18 column (4.6 mm x 100 mm, 5 µm, 1 mL/min
flow rate) and acetonitrile/water as the mobile phase with 0.1% TFA as a modifier. The gradient
started at 2% acetonitrile and increased linearly to 30% acetonitrile over 7 min, followed by a
linear increase to 80% acetonitrile by 12 min and a hold at 80% for 16 min. Purity was
determined at 254 nm and was found to be > 95%. Library compounds were purified by reverse
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phase chromatography on a preparative LC/UV/MS system (High Throughput Purification)
using a massꢀtriggered fractionation method. Compounds were eluted from the HPLC column
(MACCEL 120ꢀ10ꢀC18 SH, 10 µm, 20 mm ID x 50 mm) at 88 mL/min with an
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1
acetonitrile/water gradient using 0.1% TFA as a modifier. Nuclear magnetic resonance ( H
NMR) spectra were recorded on a Varian Mercury Plus NMR spectrometer operating at 400.13
1
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15
MHz for H using a 5 mm ASW PFG probe capable of detecting H, C, P, and N nuclei.
The proton chemical shifts (ppm) were in reference to the tetramethylsilane standard (0 ppm).
The NMR data are reported with these descriptions: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; and br, broad peak.
3
-Nitro-N-(p-tolyl)pyridin-2-amine (2a). 2ꢀChloroꢀ3ꢀnitropyridine 1 (100 mg, 0.63 mmol)
was dissolved in dioxane (4 mL) in a roundꢀbottom flask. pꢀTolylaniline (68 mg, 0.63 mmol)
was then added, followed by DIEA (0.22 mL) addition. The reaction mixture was heated to 80°C
for 24 h, and the reaction was stopped when complete as observed by LCMS. After cooling to
room temperature, the solvent was removed under reduced pressure to afford an oily residue. The
residue was dissolved in ethyl acetate (20 mL) and washed with water and brine (5 mL each).
The organic phase was separated and dried over Na SO . After filtration, the solvent was
2
4
removed under reduced pressure. The crude product was carried on to the next step without
+
further purification. MS m/z 230 [M+H] .
N-(4-(tert-Butyl)phenyl)-3-nitropyridin-2-amine (2b). 2b was prepared from 1 as described
for 2a using 4ꢀ(tertꢀbutyl)aniline. The crude product was purified by column chromatography
1
(
1% MeOH/DCM, silica gel) to give 2b (1.55 g, 89 %) as a red solid. Mp 156ꢀ158 ºC; H NMR
(400 MHz, DMSOꢀd6)
δ
9.92 (s, 1H), 8.51 (dd, J =14.5, 5.6 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),
+
7
.38 (d, J = 8.5 Hz, 1H), 6.96 (dd, J = 8.1, 4.6 Hz, 1H), 1.29 (s, 9H); MS m/z 272 [M+H] .
1
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tert-Butyl (4-((3-nitropyridin-2-yl)amino)benzyl)carbamate (2c). 2c was prepared from 1 as
described for 2a using tertꢀbutyl (4ꢀaminobenzyl)carbamate. The crude product was purified by
+
column chromatography (1% MeOH/DCM, silica gel). MS m/z 345 [M+H] .
0
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tert-Butyl (4-((4-methyl-3-nitropyridin-2-yl)amino)benzyl)carbamate (3a). 3a was prepared
from 2ꢀchloroꢀ4ꢀmethylꢀ3ꢀnitropyridine (1b) as described for 2a using tertꢀbutyl (4ꢀ
aminobenzyl)carbamate. The crude product was used directly in the next step without
+
purification. MS m/z 359 [M+H] .
tert-Butyl (4-((5-methyl-3-nitropyridin-2-yl)amino)benzyl)carbamate (3b). 3b was prepared
from 2ꢀchloroꢀ5ꢀmethylꢀ3ꢀnitropyridine (1c) as described for 2a using tertꢀbutyl (4ꢀ
aminobenzyl)carbamate. The crude product was used directly in the next step without
+
purification. MS m/z 359 [M+H] .
tert-Butyl (4-((5,6-dimethyl-3-nitropyridin-2-yl)amino)benzyl)carbamate (3c). 3c was
prepared from 2ꢀchloroꢀ5,6ꢀdimethylꢀ3ꢀnitropyridine (1d) as described for 2a using tertꢀbutyl (4ꢀ
aminobenzyl)carbamate. The crude product was used directly in the next step without
+
purification. MS m/z 373 [M+H] .
tert-Butyl (4-((6-chloro-3-nitropyridin-2-yl)amino)benzyl)carbamate (3d). 3d was prepared
from 2,6ꢀdichloroꢀ3ꢀnitropyridine (1e) as described for 2a using tertꢀbutyl (4ꢀaminobenzyl)
carbamate. The crude product was used directly in the next step without purification. MS m/z
+
378 [M+H] .
tert-Butyl (4-((5-bromo-3-nitropyridin-2-yl)amino)benzyl)carbamate (3e). 3e was prepared
from 5ꢀbromoꢀ2ꢀchloroꢀ3ꢀnitropyridine (1f) as described for 2a using tertꢀbutyl (4ꢀaminobenzyl)
carbamate. The crude product was used directly in the next step without purification. Mp 178ꢀ
1
1
80 ºC; H NMR (400 MHz, DMSOꢀd ) δ 9.95 (s, 1H), 8.65 (m, 1H), 8.54 (s, 1H), 7.51 (d, J =
6
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.4 Hz, 2H), 7.23 (d, J = 6.4 Hz, 2H), 4.21ꢀ4.12 (m, 2H), 1.41 (s, 9H); MS m/z 367, 369 [M(ꢀtꢀ
+
Bu)+H, M(ꢀtꢀBu)+2+H] .
3
-(3-(p-Tolyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (4a). Crude 3ꢀnitroꢀNꢀ(pꢀ
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tolyl)pyridinꢀ2ꢀamine 2a was dissolved in DMSO (4 mL) and MeOH (2 mL) in a round bottom
flask. 2ꢀaminonicotinaldehyde 4 (77 mg, 1.1 equivalent) and Na S O (274 mg) were added to
2
2
4
the flask. The reaction mixture was heated to 100°C for 18 h. After cooling to room temperature,
the reaction mixture was diluted with dichloromethane (50 mL) and washed with water and
brine. The organic phase was separated and dried over Na SO . After filtration, the solvent was
2
4
removed under reduced pressure. The crude product was purified by silica gel chromatography
(EtOAc/hexane) to afford 20 mg of 3ꢀ(3ꢀ(pꢀtolyl)ꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀ
1
amine (4a) as an offꢀwhite solid. Mp 186ꢀ188 °C; H NMR (400 MHz, DMSOꢀd ) δ: 8.29ꢀ8.28
6
(dd, J = 5.0, 1.6 Hz, 1H), 8.18ꢀ8.15 (dd, J = 7.8, 1.1 Hz, 1H), 7.96ꢀ7.95 (dd, J = 4.7, 1.6 Hz, 1H),
7.37ꢀ7.33 (dd, J = 8.21, 5.09 Hz, 1H), 7.31ꢀ7.26 (m, 4H), 7.20ꢀ7.17 (dd, J = 7.8, 1.9 Hz, 1H),
+
6
.96 (s, 2H), 6.39ꢀ6.36 (dd, J = 7.8, 4.6 Hz, 1H), 2.36 (s, 3H); MS m/z 302 [M+H] .
3
-(3-(4-(tert-Butyl)phenyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (4b). 4b was
1
prepared as described for 4a using 2b and 2ꢀaminonicotinaldehyde. Mp 175ꢀ180 ºC; H NMR
(400M Hz, Acetoneꢀd6) δ 8.44−8.42 (dd, J = 4.7, 1.2 Hz, 1H), 8.26ꢀ8.23 (dd, J = 8.2,1.6 Hz, 1H),
8.05ꢀ8.02 (dd, J = 6.3, 1.6 Hz, 1H), 7.75ꢀ7.73 (dd, J = 7.8, 1.6 Hz, 1H), 7.70ꢀ7.68 (dd, J = 6.6, 2.3
Hz, 2H), 7.57ꢀ7.55 (dd, J = 8.6, 1.9 Hz, 2H), 7.48ꢀ7.44 (dd, J = 8.2, 4.7 Hz, 1H), 6.82ꢀ6.78 (dd, J
+
=
7.8, 6.3 Hz, 1H), 1.41 (s, 9H). MS m/z 344 [M+H] ; Anal. (C H N ·1.5 TFA·0.1 acetone)
21
21
5
calcd: C, 56.10; H, 4.48; N 13.46. Found: C, 56.32; H, 4.25; N, 13.70.
tert-Butyl (4-(2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)carbamate
4c). 4c was prepared as described for 4a using 2c and 2ꢀaminonicotinaldehyde. Mp 172ꢀ173 ºC;
(
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H NMR (400 MHz, DMSOꢀd ) δ 8.31 (dd, J = 4.8, 1.5 Hz, 1H), 8.20 (dd, J = 8.0, 1.5 Hz, 1H),
6
7
.99 (dd, J = 4.8, 1.8 Hz, 1H), 7.49 (t, J = 6.3 Hz, 1H), 7.43 – 7.32 (m, 5H), 7.20 (dd, J = 7.7, 1.9
Hz, 1H), 6.99 (s, 2H), 6.39 (dd, J = 7.7, 4.8 Hz, 1H), 4.21 (d, J = 6.2 Hz, 2H), 1.41 (s, 9H); MS
0
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+
.
m/z 417 [M+H]
tert-Butyl
(4-(2-(2-aminopyridin-3-yl)-7-methyl-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)
1
carbamate (5a). 5a was prepared from 3a as described for 4a. Mp 160ꢀ162 ºC; H NMR (400
MHz, CD OD) δ: 8.17 (d, J = 5.1 Hz, 1H), 7.96 (dt, J = 5.1, 1.4 Hz, 1H), 7.45 (d, J = 7.8 Hz,
3
2
H), 7.37ꢀ7.31 (m, 3H), 7.24 (d, J = 5.1 Hz, 1H), 6.49ꢀ6.45 (m, 1H), 4.31 (br.s, 2H), 2.74 (s,
+
3
H), 1.46 (s, 9H); MS m/z 431 [M+H] .
tert-Butyl
(4-(2-(2-aminopyridin-3-yl)-6-methyl-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)
1
carbamate (5b). 5b was prepared from 3b as described for 4a. Mp 211ꢀ212 ºC; H NMR (400
MHz, DMSOꢀd ) δ: 8.16 (dd, J = 2.0, 0.8 Hz, 1H), 8.04–7.95 (m, 2H), 7.49 (t, J = 6.2 Hz, 1H),
6
7
.36 (s, 4H), 7.17 (dd, J = 7.7, 1.9 Hz, 1H), 7.02 (s, 2H), 6.38 (dd, J = 7.7, 4.8 Hz, 1H), 4.21 (d, J
+
=
6.2 Hz, 2H), 2.45 (d, J = 0.8 Hz, 3H), 1.41 (s, 9H), 1.34; MS m/z 431 [M+H] .
tert-Butyl (4-(2-(2-aminopyridin-3-yl)-5,6-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)
carbamate (5c). 5c was prepared from 3c as described for 4a. The crude product was used
+
without purification. MS m/z 445 [M+H] .
tert-Butyl
(4-(2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)
1
carbamate (5d). 5d was prepared from 3d as described for 4a. Mp 235ꢀ237 ºC; H NMR (400
MHz, DMSOꢀd ) δ: 8.26 (d, J = 8.6 Hz, 1H), 7.99 (dd, J = 4.7, 2.0, 1H), 7.49 (t, J = 6.1 Hz, 1H),
6
7
.45 (d, J = 8.2 Hz, 1H), 7.42ꢀ7.35 (m, 4H), 7.21 (dd, J = 7.8, 2.0 Hz, 1H), 6.92 (s, 2H), 6.41 (dd,
+
J = 7.4, 4.7 Hz, 1H), 4.22 (d, J = 6.1 Hz, 2H), 1.41 (s, 9H); MS m/z 451 [M+H] .
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tert-Butyl
4-(2-(2-aminopyridin-3-yl)-6-bromo-3H-imidazo[4,5-b]pyridine-3-yl)benzyl
carbamate (5e). 5e was prepared from 3e as described for 4a. The product was purified by
column chromatography (1ꢀ5% methanol in dichloromethane) followed by reꢀcrystallization in
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:1 ethyl acetate/hexanes. Mp 176ꢀ178 °C; H NMR (400 MHz, DMSOꢀd ) δ 8.50 (d, J = 2.0
6
Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H), 7.99 (dd, J = 4.7, 2.0 Hz, 1H), 7.48 (t, J = 5.6 Hz, 1H), 7.40ꢀ
7
.31 (m, 3H), 7.23 (dd, J = 7.8, 2.0 Hz, 1H), 6.97 (s, 2H), 6.40 (dd, J = 7.7, 4.4 Hz, 1H), 4.121
+
(
d, J = 6.2 Hz, 2H), 1.41 (s, 9H). MS m/z 495, 497 [M+H, M+2+H] .
3-(3-(4-(Aminomethyl)phenyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (6). HCl (4
M in dioxane, 7.2 mL, 28.8 mmol) was added to a solution of 4c (0.7 g, 1.7 mmol) in methanol
5 mL), and the mixture was stirred at room temperature for 4 h. The solution was concentrated
(
under reduced pressure to yield 6 (0.82 g, quantitative yield) as a hydrochloric acid salt. Mp 255ꢀ
1
2
57 ºC; H NMR (400 MHz, DMSOꢀd ) δ 8.44 (m, 3H), 8.30 (dd, J = 8.0, 1.4 Hz, 1H), 8.12 (dd,
6
J = 6.2, 1.6 Hz, 1H), 7.87 (d, J = 6.6 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H),
7
.48 (dd, J = 8.0, 4.7 Hz, 1H), 6.85 (dd, J = 7.1, 6.6 Hz, 1H), 4.12 (d, J = 5.6 Hz, 2H), 3.81 (s,
+
2
H). MS m/z 317 [M+H] Anal. (C H N ·4.6 HCl·4.2 H O·0.8 Et O) calcd: C, 41.13; H, 6.02;
;
18 16
6
2
2
N, 13.58. Found: C, 41.12; H, 6.02; N, 13.58.
3
-(3-(4-(Aminomethyl)phenyl)-7-methyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine
1
hydrochloride (7a). 7a was prepared from 5a as described for 6. Mp 210ꢀ212 ºC; H NMR (400
MHz, DMSOꢀd ) δ: 8.52 (s, 3H), 8.45 (s, 2H), 8.27 (d, J = 4.7 Hz, 1H), 8.14 (dd, J = 6.3, 1.6 Hz,
6
1
H), 7.91 (dd, J = 7.4, 1.6 Hz, 1H), 7.67–7.64 (m, 2H), 7.57–7.53 (m, 2H), 7.31 (d, J = 5.4 Hz,
+
1H), 6.88 (dd, J = 7.4, 6.2 Hz, 1H), 4.10 (m, 2H), 2.70 (s, 3H); MS m/z 331 [M+H] .
3
-(3-(4-(Aminomethyl)phenyl)-6-methyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine
1
hydrochloride (7b). 7b was prepared from 5b as described for 6. Mp 220ꢀ230 ºC; H NMR (400
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MHz, DMSOꢀd ) δ: 8.65 (s, 3H), 8.53 (s, 2H), 8.29 (dd, J = 2.0, 0.7 Hz, 1H), 8.16 (dd, J = 6.2,
6
1.7 Hz, 1H), 8.12 (dd, J = 2.0, 0.9 Hz, 1H), 7.91 (dd, J = 7.5, 1.6 Hz, 1H), 7.75–7.64 (m, 2H),
7.62–7.48 (m, 2H), 6.90 (dd, J = 7.5, 6.2 Hz, 1H), 4.10 (q, J = 5.8 Hz, 2H), 2.49 (s, 3H); MS m/z
0
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3
4
5
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8
9
0
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2
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7
8
9
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
3
31 [M+H] ; Anal. (C H N ·3.43 HCl·0.43 EtOAc) calcd: C, 50.45; H, 5.08; N, 17.04. Found:
19
18
6
C, 50.42; H, 5.21; N, 17.07.
-(3-(4-(Aminomethyl)phenyl)-5,6-dimethyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-
3
1
amine hydrochloride (7c). 7c was prepared from 5c as described for 6. Mp 103ꢀ112 ºC; H
NMR (400 MHz, DMSOꢀd ) δ: 7.91 (s, 2H), 7.45ꢀ7.40 (m, 2H), 7.32ꢀ7.26 (m, 2H), 7.11 (d, J =
6
7.2 Hz, 1H), 6.99 (m, 2H), 6.38 (dd, J = 7.2, 6.3 Hz, 1H), 3.80 (s, 2H), 3.40ꢀ3.15 (br.s, 3H), 2.41
+
(s, 3H), 2.35 (s, 3H); MS m/z 345 [M+H] .
3
-(3-(4-(Aminomethyl)phenyl)-5-chloro-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine
1
hydrochloride (7d). 7d was prepared from 5d as described for 6. Mp 208ꢀ211 ºC; H NMR (400
MHz, DMSOꢀd ) δ: 8.55 (s, 3H), 8.37 (dd, J = 8.4, 1.0 Hz, 1H), 8.18 – 8.12 (m, 1H), 7.97 – 7.91
6
(m, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.61 – 7.51 (m, 3H), 6.90 (t, J = 6.9 Hz, 1H), 4.11 (d, J = 5.9
+
Hz, 2H), 3.57 (s, 2H); MS m/z 351 [M+H] ; Anal. (C H N Cl·2.58 HCl·0.16 EtOAc) calcd: C,
1
8
15
6
48.78; H, 4.14; N, 18.31. Found: C, 48.80; H, 4.32; N, 18.31.
3
-(3-(4-(Aminomethyl)phenyl)-6-bromo-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine
o
1
hydrochloride (7e). 7e was prepared from 5e as described for 6. Mp. 221ꢀ223 C; H NMR
(
DMSOꢀd ) 400 M Hz δ: 8.60 (d, J = 2.0 Hz, 1H), 8.56 (bs, 2H), 8.51 (d, J = 2.4 Hz, 1H), 8.38
6
(bs, 2H), 8.13 (dd, J = 2.0 Hz and 4.7 Hz, 1H), 7.95 (dd, J = 1.6 Hz and 7.4 Hz, 1H), 7.63 (d, J =
8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 6.89 (dd, J = 6.2 Hz and 7.4 Hz, 1H), 4.17 (app q, J = 5.5
+
Hz, 1H); MS m/z 394, 396 [M+H, M+2+H] .
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tert-Butyl
(4-(2-(2-aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)
carbamate (8a). tertꢀButyl (4ꢀ(2ꢀ(2ꢀaminopyridinꢀ3ꢀyl)ꢀ5ꢀchloroꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ3ꢀ
yl)benzyl)carbamate (5d) (5.1 g, 11.33 mmol) and phenyl boronic acid (2.76 g, 22.6 mmol) in
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suspension (200 mL toluene/200 mL EtOH) were treated with saturated aqueous NaHCO (33
3
mL), and the mixture was degassed for 5 min. The catalyst Pd(PPh ) (0.51 g, 0.44 mmol) was
3
4
added, and the reaction mixture was stirred overnight at 100 °C. Upon cooling to room
temperature, the mixture was extracted with DCM (3 x 100 mL). The combined organic extracts
were washed with sat. aq. NaHCO and then with water. The solution was dried over Na SO ,
3
2
4
filtered and concentrated in vacuo. The crude product was purified by column chromatography
hexanes/ethyl acetate, 5:95). The tertꢀbutyl (4ꢀ(2ꢀ(2ꢀaminopyridinꢀ3ꢀyl)ꢀ5ꢀphenylꢀ3Hꢀ
(
imidazo[4,5ꢀb]pyridinꢀ3ꢀyl)benzyl)carbamate (8a) was obtained as dark red crystals (4.7 g,
1
8
4%). Mp 209ꢀ212 ºC; H NMR (400 MHz, DMSOꢀd ) δ: 8.25 (dd, J = 8.4, 2.3 Hz, 1H), 8.04 –
6
7.94 (m, 4H), 7.50 – 7.33 (m, 8H), 7.20 (dd, J = 7.8, 1.9 Hz, 1H), 7.00 (s, 2H), 6.40 (dd, J = 7.6,
+
4
.9 Hz, 1H), 4.22 (d, J = 6.2 Hz, 2H), 1.39 (s, 9H); MS m/z 493 [M+H] .
tert-Butyl (4-(5-(3-acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)benzyl)carbamate (8b). 8b was prepared from 5d as described for 8a using (3ꢀ
acetamidophenyl)boronic acid. The crude product was purified by column chromatography (95:5
+
ethyl acetate/methanol). MS m/z 550 [M+H] .
tert-Butyl (4-(5-(4-acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)benzyl)carbamate (8c). 8c was prepared from 5d as described for 8a, using (4ꢀ
acetamidophenyl)boronic acid. The crude product was purified by column chromatography (5:95
+
hexanes/ethyl acetate). MS m/z 550 [M+H] .
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tert-Butyl (4-(2-(2-aminopyridin-3-yl)-5-(1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-3-yl)
benzyl)carbamate (8d). 8d was prepared from 5d as described for 8a using (1Hꢀpyrazolꢀ4ꢀ
1
yl)boronic acid. Mp. 243ꢀ244 °C; H NMR (400 MHz, DMSOꢀd ) δ 12,99 (s, 1H), 8.19 (s, 1H),
6
0
1
2
3
4
5
6
7
8
9
0
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2
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.12 (d, J = 8.8 Hz, 1H), 7.95 (d, J = 5.2 Hz, 1H), 7.92 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.47 (t,
J = 5.6 Hz, 1H), 7.40ꢀ7.33 (m, 4H), 7.13 (d, J = 7.2 Hz, 1H), 6.94 (s, 2H), 6.36 (t, J = 6.0 Hz,
+
1
H), 4.20 (d, J = 6.4Hz, 2H), 1.39 (s, 9H); MS m/z 483 [M+H] .
tert- Butyl (4-(2-(2-aminopyridin-3-yl)-6-(pyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-yl)
benzyl)carbamate (8e). 8e was prepared from 5e as described for 8a using (pyridinꢀ3ꢀyl)boronic
acid. The crude residue was purified by silica gel chromatography (2ꢀ20% methanol in ethyl
1
acetate), yielding 3.9 g of 8e (7.9 mmol, 99% yield). H NMR (400 MHz, DMSOꢀd ) δ: 8.98 (d,
6
J = 1.9 Hz, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.59 (dd, J = 1.6 Hz and 4.7 Hz, 1H), 8.54 (d, J = 2.0
Hz, 1H), 8.20 (m, 1H), 7.98 (m, 1H), 7.52 (m, 2H), 7.48ꢀ7.34 (m, 4H), 7.23 (m, 1H), 7.01 (s, br,
+
2
H), 6.38 (m, 1H), 4.19 (m, 2H), 1.38 (s, 9H); MS m/z 438, 494, [Mꢀ(tꢀBu)+H, M+H] .
3
-(3-(4-(Aminomethyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine
1
hydrochloride (9a). 9a was prepared from 8a as described for 6. Mp 215ꢀ218 ºC; H NMR (400
MHz, DMSOꢀd ) δ: 8.52 (s, 3H), 8.35 (d, J = 8.2 Hz, 1H), 8.16 – 8.11 (m, 1H), 8.05 (t, J = 8.5
6
Hz, 3H), 7.90 (d, J = 7.0 Hz, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.65 – 7.59 (m, 2H), 7.50 – 7.37 (m,
+
3
H), 6.88 (q, J = 5.7, 4.9 Hz, 1H), 4.68 (s, 2H), 4.11 (d, J = 5.9 Hz, 2H); MS m/z 393 [M+H] ;
Anal. (C H N ·3.14 HCl ·0.45 EtOAc) calcd: C, 56.69; H, 4.93; N, 15.38. Found: C, 56.68; H,
2
4
20
6
5.18; N, 15.37.
N-(3-(3-(4-(Aminomethyl)phenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-
yl)phenyl)acetamide hydrochloride (9b). 9b was prepared from 8b as described for 6. Mp 245ꢀ
1
248 ºC; H NMR (400 MHz, DMSOꢀd ) δ: 10.16 (s, 1H), 8.47 (s, 3H), 8.39 (s, 2H), 8.35 (d, J =
6
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.1 Hz, 1H), 8.18 (d, J = 2.6 Hz, 1H), 8.15 – 8.08 (m, 1H), 7.89 (d, J = 8.1 Hz, 2H), 7.63 (dt, J =
11.8, 7.5 Hz, 5H), 7.36 (t, J = 7.9 Hz, 1H), 6.87 (t, J = 6.6 Hz, 1H), 4.10 (d, J = 5.9 Hz, 2H), 2.04
+
(s, 3H); MS m/z 450 [M+H] ; Anal. (C H N O ·3.35 HCl) calcd: C, 54.63; H, 4.65; N, 17.15.
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23
7
10
11
12
13
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15
16
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41
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Found: C, 54.62; H, 4.67; N, 16.81.
N-(4-(3-(4-(Aminomethyl)phenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-
yl)phenyl)acetamide hydrochloride (9c). 9c was prepared from 8c as described for 6. Mp 227ꢀ
1
2
30 ºC; H NMR (400 MHz, DMSOꢀd ) δ: 10.25 (s, 1H), 8.58 (s, 3H), 8.33 (d, J = 8.4 Hz, 1H),
6
8.16 (dd, J = 6.4, 1.7 Hz, 1H), 8.01 (t, J = 7.9 Hz, 3H), 7.90 (dd, J = 7.6, 1.7 Hz, 1H), 7.75 – 7.59
(m, 6H), 6.90 (t, J = 6.9 Hz, 1H), 5.80 (s, 2H), 4.13 (d, J = 5.8 Hz, 2H), 2.07 (s, 3H); MS m/z 450
+
.
.
[M+H] ; Anal. (C H N O ·3.35 HCl 0.28 EtOAc 0.3 H O) calcd: C, 54.33; H, 4.90; N, 16.35.
26
23
7
2
Found: C, 54.35; H, 4.96; N, 16.11.
-(3-(4-(Aminomethyl)phenyl)-5-(1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-
3
yl)pyridin-2-amine hydrochloride (9d). 9d was prepared from 8d as described for 6. Mp. 264ꢀ
1
2
69 °C; H NMR (400 MHz, DMSOꢀd ) δ: 8.50 (br, 4H), 8.43 (br, 1H), 8.22 (d, J = 8.4 Hz, 1H),
6
8.13ꢀ8.09 (m, 3H), 7.85 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.0 Hz, 2H),
+
7.58 (d, J = 8.8 Hz, 2H), 6.86 (t, J = 7.2 Hz, 1H), 4.10 (q, J = 5.2 Hz, 2H); MS m/z 383 [M+H] .
3
-(3-(4-(Aminomethyl)phenyl)-6-(pyridin-3-yl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-
1
amine hydrochloride (9e). 9e was prepared from 8e as described for 6. H NMR (400 MHz,
(
CD OD) δ: 9.38 (d, J = 2.0 Hz, 1H), 9.11 (d, J = 2.0 Hz, 1H), 8.95ꢀ8.93 (m, 1H), 8.89 (d, J =
3
2.0 Hz, 1H), 8.78 (m, 1H), 8.83ꢀ8.25 (m, 1H), 8.06ꢀ8.04 (m, 1H), 7.88ꢀ7.86 (m, 1H), 7.77ꢀ7.66
+
(m, 4H), 6.86ꢀ6.83 (m, 1H), 4.23 (m, 2H), MS m/z 394 [M+H] .
2
-Methyl-N-(propan-2-ylidene)propane-2-sulfinamide (11). Ti(OEt) (5.36 mL) was added
4
dropwise over the course of 15 min to a solution of 2ꢀmethylꢀ2ꢀpropanesulfinamide (10) (1.47 g,
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2.1 mmol) and acetone (0.98 mL) in THF (24 mL) chilled to 0 ºC. After being stirred at room
temperature for 3 days, the reaction mixture was quenched by the rapid addition of an iceꢀcooled
saturated sodium bicarbonate solution. The suspension was filtered through glassꢀfiber filter
0
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paper and was washed with ethyl acetate. The filtrate was washed with sat. aq. NaHCO solution
3
and brine. The organics were dried over Na SO and concentrated in vacuo. Purification by
2
4
column chromatography (10ꢀ60% ethyl acetate in hexanes) yielded 2ꢀmethylꢀNꢀ(propanꢀ2ꢀ
1
ylidene)propaneꢀ2ꢀsulfinamide (11) (0.92 g, 47%). H NMR (400 MHz, CDCl ) δ: 2.34 (s, 3H),
3
2.18 (s, 3H), 1.23 (s, 9H).
N-(2-(4-Aminophenyl)propan-2-yl)-2-methylpropane-2-sulfinamide (12). A solution of nꢀ
butyllithium in hexanes (1.65 M, 5.2 mL) was added dropwise to a solution of 4ꢀbromoꢀN,Nꢀ
bis(trimethylsilyl)aniline (2.55 mL) in THF (14 mL) at ꢀ78 ºC. The resulting mixture was stirred
for 1.5 h at ꢀ78 ºC, after which a solution of Nꢀisopropylideneꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide
(11) in THF (2.8 mL) was added dropwise at ꢀ78 ºC. The reaction mixture was stirred for an
additional 1 h at ꢀ78 ºC and then allowed to warm to room temperature. After being stirred for 18
h at room temperature, the reaction mixture was poured into an iceꢀcooled sat. aq. NaHCO3
solution. The aqueous layer was extracted with ethyl acetate. The combined organic layers were
washed with a sat. aq. NaHCO solution and brine and then dried over Na SO . The dried
3
2
4
solution was filtered, and the filtrate was concentrated to dryness. The residue was diluted with
diisopropylether and extracted with a 1 M citric acid solution. The aqueous extracts were
combined and alkalinized with solid sodium bicarbonate to pH>7. The basic aqueous layer was
extracted with ethyl acetate, and the organic layer was washed with brine. The organics were
dried over anhydrous Na SO and concentrated in vacuo. Purification by column
2
4
chromatography (50ꢀ100% ethyl acetate in hexanes) produced Nꢀ(2ꢀ(4ꢀaminophenyl)propanꢀ2ꢀ
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1
yl)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide (12) (173 mg, 12%). H NMR (400 MHz, CDCl ) δ: 7.26 (d,
3
J = 8.7 Hz, 2H), 6.65 (d, J = 8.7 Hz, 2H), 3.66 (brs, 1H), 3.31 (s, 1H), 1.67 (s, 3H), 1.64 (s, 3H),
1.20 (s, 9H).
10
11
12
13
14
15
16
17
18
19
20
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22
23
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29
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N-(2-(4-((6-Chloro-3-nitropyridin-2-yl)amino)phenyl)propan-2-yl)-2-methylpropane-2-
sulfinamide (13). 13 was prepared from 2,6ꢀdichloroꢀ3ꢀnitropyridine (1e) as described for 2a
using Nꢀ(2ꢀ(4ꢀaminophenyl)propanꢀ2ꢀyl)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide (12). The crude
product was purified by column chromatography (30ꢀ100% ethyl acetate in hexanes). MS m/z
+
411 [M+H] .
tert-Butyl (2-(4-((6-chloro-3-nitropyridin-2-yl)amino)phenyl)propan-2-yl)carbamate (14).
A solution of HCl in dioxane (4 M, 130 ꢀL) was added dropwise to a solution of Nꢀ(1ꢀ(4ꢀ((6ꢀ
chloroꢀ3ꢀnitropyridinꢀ2ꢀyl)amino)phenyl)ꢀ1ꢀmethylethyl)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide (13)
(
107 mg, 0.26 mmol) in DCM (5 mL) at room temperature. The reaction mixture was stirred for
30 min. Hexane was added to the suspension, and the solid was filtered to yield an Nꢀ(4ꢀ(1ꢀ
aminoꢀ1ꢀmethylethyl)phenyl)ꢀ6ꢀchloroꢀ3ꢀnitropyridinꢀ2ꢀamine hydrochloride intermediate. Sat.
aq. NaHCO solution (6 mL) was added to a suspension of Nꢀ(4ꢀ(1ꢀaminoꢀ1ꢀ
3
methylethyl)phenyl)ꢀ6ꢀchloroꢀ3ꢀnitropyridinꢀ2ꢀamine hydrochloride in THF (3 mL) chilled to 0
ºC, followed by dropwise addition of a solution of diꢀtertꢀbutyl dicarbonate (167 mg) in THF (3
mL). After being stirred at room temperature overnight, the reaction mixture was extracted with
ethyl acetate. The organic layer was washed with sat. aq. NaHCO solution and brine. The
3
organics were dried over Na SO and concentrated in vacuo. Purification by column
2
4
chromatography (3ꢀ30% ethyl acetate in hexanes) produced tertꢀbutyl (2ꢀ(4ꢀ((6ꢀchloroꢀ3ꢀ
1
nitropyridinꢀ2ꢀyl)amino)phenyl) propanꢀ2ꢀyl)carbamate (14) (99 mg, 2 steps 93%). H NMR
(
400 MHz, CDCl ) δ: 10.25 (br.s, 1H), 8.46 (d, J = 8.7Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.43 (d,
3
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J = 8.7 Hz, 2H), 6.79 (d, J = 8.7 Hz, 1H), 4.94 (brs, 1H), 1.64 (s, 6H), 1.50ꢀ1.00 (m, 9H); MS
m/z 351 [Mꢀ(tꢀBu)+H].
tert-Butyl (2-(4-(2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)
propan-2-yl)carbamate (15). 15 was prepared as described for 4a using 14 and 2ꢀ
aminonicotinaldehyde. Purification by column chromatography (10ꢀ100% ethyl acetate in
0
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5
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7
8
9
0
1
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3
4
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hexanes) produced tertꢀbutyl (2ꢀ(4ꢀ(2ꢀ(2ꢀaminopyridinꢀ3ꢀyl)ꢀ5ꢀchloroꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ
+
3
ꢀyl)phenyl)propanꢀ2ꢀyl)carbamate (15) (30 mg, 26%). MS m/z 479 [M+H] .
tert-Butyl (2-(4-(2-(2-aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)
propan-2-yl)carbamate (16). 16 was prepared as described for 8a using 15 and phenyl boronic
acid. Purification by column chromatography (30ꢀ100% ethyl acetate in hexanes) yielded tertꢀ
butyl (2ꢀ(4ꢀ(2ꢀ(2ꢀaminopyridinꢀ3ꢀyl)ꢀ5ꢀphenylꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ3ꢀyl)phenyl) propanꢀ2ꢀ
+
yl)carbamate (16) (27 mg, 81%). MS m/z 521 [M+H] .
3
-(3-(4-(2-Aminopropan-2-yl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-
1
amine (17). 17 was prepared from 16 as described for 6. H NMR (400 MHz, DMSOꢀd ) δ:
6
8.90ꢀ8.70 (m, 3H), 8.37 (d, J = 8.3 Hz, 2H), 8.15 (dd, J = 6.0 Hz and 1.8 Hz, 1H,), 8.08ꢀ8.03 (m,
3H), 7.86 (dd, J = 7.3 Hz and 1.4 Hz, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H),
7.52ꢀ7.47 (m, 2H), 7.46ꢀ7.41 (m, 1H), 6.89 (dd, J = 7.3 Hz and 6.4 Hz, 1H), 1.70 (s, 6H); MS
+
m/z 421 [M+H] .
tert-Butyl (1-(4-((6-chloro-3-nitropyridin-2-yl)amino)phenyl)cyclobutyl)carbamate (18). A
solution of 2,6ꢀdichloroꢀ3ꢀnitropyridine (1e) (5.11 g, 26.4 mmol) in DMA (50 mL) and
triethylamine (5 mL) cooled to 0 ºC was added dropwise to a solution of tertꢀbutyl (1ꢀ(4ꢀamino
phenyl)cyclobutyl)carbamate (6.3 g, 24.0 mmol)) in DMA (25 mL). The reaction mixture was
stirred at 0 ºC for one hour and then at room temperature overnight. The reaction mixture was
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then diluted with water (250 mL) and extracted with ethyl acetate (2 x 200 mL). The organics
were combined and washed with a sat. aq. NaHCO solution, water and brine. The organics were
3
dried over Na SO and concentrated in vacuo. Purification by column chromatography (15%
2
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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ethyl acetate in hexanes) produced
tertꢀbutyl (1ꢀ(4ꢀ((6ꢀchloroꢀ3ꢀnitropyridinꢀ2ꢀ
1
yl)amino)phenyl)cyclobutyl)carbamate (18) as an orange solid (5.05 g, 50%). H NMR (400
MHz, DMSOꢀd ) δ: 10.05 (s, 1H), 8.52 (d, J = 8.8Hz, 1H), 7.56 – 7.52 (m, 2H), 7.42 – 7.37 (m,
6
3H), 6.98 (d, J = 8.8 Hz, 1H), 2.47 – 2.34 (m, 4H), 2.04 – 1.96 (m, 1H), 1.84 – 1.74 (m, 1H),
+
1
.30 (bs, 9H); MS m/z 419 [M+H] .
tert-Butyl (1-(4-(2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)
cyclobutyl)carbamate (19). 19 was prepared as described for 6 using 18 and 2ꢀ
1
aminonicotinaldehyde. H NMR (400M Hz, DMSOꢀd )
δ 8.26 (d, J = 8.0 Hz, 1H), 8.00 – 7.96
6
(m, 1H), 7.69 (bs, 1H), 7.54 – 7.35 (m, 5H), 7.24 – 7.08 (m, 1H), 7.04 – 6.96 (m, 2H), 6.32 –
6.28 (m, 1H), 2.48 – 2.35 (m, 4H), 2.06 – 1.96 (m, 1H), 1.86 – 1.76 (m, 1H), 1.40 – 1.06 (m,
+
9
H); MS m/z 491 [M+H] .
tert-Butyl (1-(4-(2-(2-aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]pyridin-3-
yl)phenyl)cyclobutyl)carbamate (20a). 20a was prepared from 19 as described for 8a using
phenylboronic acid. The crude product was purified by column chromatography (10ꢀ100% ethyl
acetate in hexanes), yielding 20a with some impurities. The solid was reꢀcrystallized from ethyl
1
acetate, affording an offꢀwhite solid (7.2 g). Mp 111ꢀ116 °C; H NMR (400M Hz, DMSOꢀd ) δ:
6
8
7
2
.23 (d, J = 8.0 Hz, 1H), 8.04 – 7.98 (m, 3H), 7.94 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H),
.46 – 7.35 (m, 6H), 7.18 – 7.14 (m, 1H), 6.90 (bs, 1H), 6.33 (dd, J = 7.6Hz and 4.4 Hz, 1H),
.48 – 2.40 (m, 4H), 2.09 – 2.00 (m, 1H), 1.89 – 1.79 (m, 1H), 1.30 (m, 9H); MS m/z 533
+
[M+H] .
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tert-Butyl (1-(4-(5-(3-acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-
b]pyridin-3-yl)phenyl)cyclobutyl)carbamate (20b). 20b was prepared from 19 as described for
8
a using (3ꢀacetamidophenyl)boronic acid. Purification by column chromatography (0ꢀ10%
0
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3
4
5
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8
9
0
1
2
3
4
5
6
7
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methanol in dichloromethane) yielded 20b as a brown solid with some coꢀeluting impurities. The
fractions with the product were combined and concentrated to produce a dark brown solid. The
1
solid was triturated with ethyl acetate to afford a tan solid (2.6 g, 70%). Mp 160ꢀ163 °C; H
NMR (400M Hz, DMSOꢀd ) δ: 8.23 (d, J = 8.0 Hz, 1H), 8.04 – 7.98 (m, 3H), 7.94 (d, J = 8.0
6
Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.46 – 7.35 (m, 6H), 7.18 – 7.14 (m, 1H), 6.90 (bs, 1H), 6.33
(
dd, J = 7.6Hz and 4.4 Hz, 1H), 2.48 – 2.40 (m, 4H), 2.09 – 2.00 (m, 1H), 1.89 – 1.79 (m, 1H),
+
.
1.30 (m, 9H); MS m/z 590 [M+H] ; Anal. (C H N O 0.18 EtOAc) calcd: C, 68.87; H, 6.07;
3
4
35
7
3
N, 16.19. Found: C, 69.12; H, 6.34; N, 15.94.
tert-Butyl (1-(4-(2-(2-aminopyridin-3-yl)-5-(pyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-yl)
phenyl)cyclobutyl)carbamate (20c). 20c was prepared from 19 as described for 8a using
pyridinꢀ3ꢀylboronic acid. Purification by column chromatography (0ꢀ10% methanol in
dichloromethane) produced 20c with some impurities. The solid was triturated with ethyl acetate
1
to yield a yellow solid (0.084 g, 79%). Mp 163ꢀ164 °C; H NMR (400M Hz, DMSOꢀd ) δ: 9.21
6
(
dd, J = 2.4, 0.8 Hz, 1H), 8.56 (dd, J = 4.8, 1.6 Hz, 1H), 8.34 (ddd, J = 8.1, 2.3, 1.6 Hz, 1H), 8.29
(d, J = 8.3 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.97 (dd, J = 4.8, 1.9 Hz, 1H), 7.55 – 7.48 (m, 2H),
7
.45 (dt, J = 11.8, 4.2 Hz, 3H), 7.04 (s, 2H), 6.31 (dd, J = 7.7, 4.8 Hz, 1H),4.45ꢀ2.40 (m, 4H),
+
2
.10ꢀ1.90 (m, 1H), 1.85ꢀ1.75 (s, 1H), 1.33 (s, 9H); MS m/z 534 [M+H] .
3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-
amine hydrochloride (21a). 21a was prepared from 20a as described for 6. The product was
suspended in Et O and then filtered off, producing 3ꢀ(3ꢀ(4ꢀ(1ꢀaminocyclobutyl)phenyl)ꢀ5ꢀ
2
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phenylꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀamine hydrochloride (21a) as a white solid. Mp
1
280ꢀ285 ºC; H NMR (400 MHz, DMSOꢀd ) δ: 8.94 (s, 3H), 8.47 (bs, 1H) 8.38 (d, J = 8.8Hz,
6
1H), 8.19 – 8.15 (m, 1H), 8.10 – 8.03 (m, 3H), 7.93 – 7.88 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H),
10
11
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13
14
15
16
17
18
19
20
21
22
23
24
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29
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44
45
46
47
48
49
50
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58
59
60
7
.69 (d, J = 8.4 Hz, 2H), 7.52 – 7.40 (m, 3H), 6.92 (t, J = 7.6 Hz, 1H), 2.70 – 2.57 (m, 4H), 2.29
+
– 2.20 (m, 1H), 1.90 – 1.80 (m, 1H); MS m/z 433 [M+H] ; Anal. (C H N ·3.06 HCl·0.01
2
7
24
6
.
dioxane 0.03 diethylether) calcd: C, 59.61; H, 5.28; N, 15.36. Found: C, 59.62; H, 5.05; N,
15.36.
N-(3-(3-(4-(1-Aminocyclobutyl)phenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-
b]pyridin-5-yl)phenyl)acetamide hydrochloride (21b). 21b was prepared from 20b as
described for 6. The product was suspended in Et O and then filtered off, producing Nꢀ(3ꢀ(3ꢀ(4ꢀ
2
(1ꢀaminocyclobutyl)phenyl)ꢀ2ꢀ(2ꢀaminopyridinꢀ3ꢀyl)ꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ5ꢀ
1
yl)phenyl)acetamide hydrochloride (21b) as a yellow solid. Mp 233ꢀ237 ºC; H NMR (400 MHz,
DMSOꢀd ) δ: 10.25 (s, 1H), 8.98 (bs, 3H), 8.56–8.43 (m, 2H), 8.38 (d, J = 8.4 Hz, 1H), 8.24–
6
8
.22 (m, 1H), 8.18 (dd, J = 4.8 Hz and 1.6 Hz, 1H), 7.96–7.92 (m, 1H), 7.79–7.66 (m, 6H), 7.33
(t, J = 8.0 Hz, 1H), 6.93 (dd, J = 7.6 Hz and 6.0 Hz 1H), 2.70–2.58 (m, 4H), 2.32–2.20 (m, 1H),
+
2.08 (s, 3H), 1.92–1.82 (m, 1H); MS m/z 433 [M+H] ; Anal. (C H N O·3.36 HCl·0.23 dioxane
2
9
27
7
.
0
.06 diethylether) calcd: C, 56.89; H, 5.19; N, 15.40. Found: C, 56.89; H, 5.18; N, 15.40.
3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-(pyridin-3-yl)-3H-imidazo[4,5-b]pyridin-2-
yl)pyridin-2-amine hydrochloride (21c). 21c was prepared from 20c as described for 6. The
product was suspended in Et O and then filtered off, yielding 3ꢀ(3ꢀ(4ꢀ(1ꢀ
2
aminocyclobutyl)phenyl)ꢀ5ꢀ(pyridinꢀ3ꢀyl)ꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀamine
1
hydrochloride (21c) as a yellow solid. Mp 283ꢀ287 ºC; H NMR (400 MHz, DMSOꢀd ) δ: 9.45
6
(s, 1H), 9.10–9.00 (m, 4H), 8.90 (d, J = 4.8 Hz, 1H), 8.59–8.49 (m, 3H), 8.33 (d, J = 8.8 Hz, 1H),
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5
.20 (d, J = 6.0 Hz, 1H), 8.10–8.04 (m, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 8.4 Hz, 2H),
.71 (d, J = 8.8 Hz, 2H), 6.95 (t, J = 6.4 Hz, 1H), 2.64 (t, J = 8.0 Hz, 4H), 2.32 – 2.20 (m, 1H),
+
.92 – 1.82 (m, 1H); MS m/z 434 [M+H] ; Anal. (C H N O·3.94 HCl·0.26 dioxane) calcd: C,
2
6
23
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
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7
8
9
0
1
2
3
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6
7
8
9
0
4.12; H, 4.87; N, 16.34. Found: C, 54.12; H, 5.11; N, 16.33.
N-(1-(4-(2-(2-Aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]pyridin-3-
yl)phenyl)cyclobutyl)acetamide (22). 3ꢀ(3ꢀ(4ꢀ(1ꢀaminocyclobutyl)phenyl)ꢀ5ꢀphenylꢀ3Hꢀ
imidazo[4,5ꢀb]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀamine hydrochloride (21a) (22 mg, 0.05 mmol) was
dissolved in pyridine (1.0 mL), followed by the addition of acetic anhydride (4.7 ꢀL, 0.05
mmol). The mixture was stirred overnight at room temperature, diluted with DCM/DMF (1:1, 2
mL) and then stirred for an additional 12 h. The mixture was diluted with DMSO, filtered and
purified by HPLC to afford Nꢀ(1ꢀ(4ꢀ(2ꢀ(2ꢀaminopyridinꢀ3ꢀyl)ꢀ5ꢀphenylꢀ3Hꢀimidazo[4,5ꢀ
b]pyridinꢀ3ꢀyl)phenyl)cyclobutyl)acetamide (22) (14 mg, 57%) as a white powder after freeze
drying. NMR (400 MHz, DMSOꢀd ) δ: 8.53 (1H, s), 8.27 (1H, d, J = 8.6 Hz), 8.03 (2H, d, J =
6
7.5 Hz), 8.00 (1H, dd, J = 5.2, 1.7 Hz), 7.98 (1H, d, J = 8.0 Hz), 7.54 (2H, d, J = 8.6 Hz), 7.48
(2H, t, J = 7.5 Hz), 7.43 (2H, d, J = 8.6 Hz), 7.40 (1H, t, J = 8.6 Hz), 7.13 (1H, dd, J = 8.0, 1.7
Hz), 6.99 (2H, s), 6.38 (1H, dd, J = 7. 5, 4.6 Hz), 2.51ꢀ2.45 (4H, m), 2.09ꢀ2.00 (1H, m), 1.92ꢀ
+
1.83 (1H, m); MS m/z 475 [M+H] .
3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-(3-aminophenyl)-3H-imidazo[4,5-b]pyridin-2-
yl)pyridin-2-amine hydrochloride (23). HCl in dioxane (4.0 M, 1 mL) was added to a solution
of Nꢀ(3ꢀ(3ꢀ(4ꢀ(1ꢀaminocyclobutyl) phenyl)ꢀ2ꢀ(2ꢀaminopyridinꢀ3ꢀyl)ꢀ3Hꢀimidazo[4,5ꢀb]pyridinꢀ
5ꢀyl)phenyl) acetamide (21b) (0.180 g, 0.42 mmol) in dichloromethane (5 mL) and methanol (1
mL). The reaction was capped and heated at 50ºC for 3 days. Upon completion, the reaction was
diluted with ether (20 mL), and 3ꢀ(3ꢀ(4ꢀ(1ꢀaminocyclobutyl)phenyl)ꢀ5ꢀ(3ꢀaminophenyl)ꢀ3Hꢀ
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imidazo[4,5ꢀb]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀamine hydrochloride (23) was filtered from the mixture as
1
a yellow solid (0.162 g, 87%). Mp decomposed at 240 ºC; H NMR (400 MHz, DMSOꢀd ) δ:
6
9
.96 (bs, 3H), 8.44–8.30 (m, 3H), 8.16 (bs, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.01–8.79 (m, 2H),
.89 (d, J = 5.2 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0Hz, 2H), 7.57–7.50 (m, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
7.32–7.28 (m, 1H), 6.94–6.86 (m, 1H), 2.72–2.58 (m, 4H), 2.32–2.20 (m, 1H), 1.92–1.82 (m,
+
.
1
H); MS m/z 448 [M+H] ; Anal. (C H N ·3.73 HCl·0.17 dioxane 0.73 CH Cl ) calcd: C,
27 25 7 2 2
51.66; H, 4.81; N, 14.84. Found: C, 51.66; H, 4.81; N, 14.85.
Methyl 1-(4-((6-chloro-3-nitropyridin-2-yl)amino)phenyl)cyclobutane-1-carboxylate (24).
24 was prepared as described for 2a using 1e and methyl 1ꢀ(4ꢀaminophenyl)cyclobutaneꢀ1ꢀ
1
carboxylate. H NMR (400 MHz, CDCl ) δ: 10.27 (s, 1H), 8.47 (dd, J = 8.6, 1.7 Hz, 1H), 7.63
3
(dd, J = 8.6, 1.1 Hz, 2H), 7.35 (dd, J = 8.6, 1.1 Hz, 2H), 6.81 (dd, J = 8.6, 1.1 Hz, 1H), 3.67 (d, J
=
1.7 Hz, 3H), 2.88ꢀ2.83 (m, 2H), 2.56ꢀ2.49 (m, 2H), 2.10ꢀ2.00 (m, 1H), 1.94ꢀ1.85 (m, 1H); MS
+
m/z 362 [M+H] .
Methyl 1-(4-((3-nitro-6-phenylpyridin-2-yl)amino)phenyl)cyclobutane-1-carboxylate (25).
1
25 was prepared as described for 8a using 24 and phenylboronic acid. H NMR (400 MHz,
CDCl ) δ: 10.33 (s, 1H), 8.59 (d, J = 8.7 Hz, 1H), 8.08ꢀ8.03 (m, 2H), 7.76 (dt, J = 9.2, 2.4 Hz,
3
2H), 7.53ꢀ7.49 (m, 3H), 7.36 (dt, J = 8.9, 2.3 Hz, 2H), 7.31 (d, J = 8.7 Hz, 1H), 3.68 (s, 3H),
2.90ꢀ2.83 (m, 2H), 2.60ꢀ2.51 (m, 2H), 2.12ꢀ2.00 (m, 1H), 1.97ꢀ1.86 (m, 1H); MS m/z 404
+
[
M+H] .
Methyl
1-(4-(2-(2-aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)
cyclobutane-1-carboxylate (26). 26 was prepared as described for 4a using 25 and 2ꢀ
1
aminonicotinaldehyde. H NMR (400 MHz, DMSOꢀd ) δ: 8.27 (d, J = 8.6 Hz, 1H), 8.04–8.02
6
(m, 2H), 8.01 (dd, J = 4.9, 2.0 Hz, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.57–7.50 (m, 1H), 7.50–7.44
29
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