Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening

Article abstract of DOI:10.1021/acs.jmedchem.9b01670

The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize N-acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET-KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.

Full text of DOI:10.1021/acs.jmedchem.9b01670

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Discovery of a Bromodomain and Extraterminal Inhibitor with a Low
Predicted Human Dose through Synergistic Use of Encoded Library
Technology and Fragment Screening
Christopher R. Wellaway,*,† Dominique Amans,† Paul Bamborough,† Heather Barnett,† Rino A. Bit,†
Jack A. Brown,† Neil R. Carlson,‡ Chun-wa Chung,† Anthony W. J. Cooper,† Peter †D. Craggs,†
Robert P. Davis,† Tony W. Dean,† John P. Evans,† Laurie Gordon,† Isobel L. Harada, David J. Hirst,†
Philip G. Humphreys,† Katherine L. Jones,† Antonia J. Lewis,† Matthew J. Lindon,†,§ Dave Lugo,†
Mahnoor Mahmood,† Scott McCleary,† Patricia Medeiros,‡ Darren J. Mitchell,† Michael O’Sullivan,†
Armelle Le Gall,† Vipulkumar K. Patel,† Chris Patten,† Darren L. Poole,† Rishi R. Shah,†
Jane E. Smith,† Kayleigh A. J. Stafford,† Pamela J. Thomas,† Mythily Vimal,† Ian D. Wall,†
Robert J. Watson,† Natalie Wellaway,† Gang Yao,‡ and Rab K. Prinjha†
†GSK, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.
‡GSK, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140, United States
S
* Supporting Information
ABSTRACT: The bromodomain and extraterminal (BET)
family of bromodomain-containing proteins are important
regulators of the epigenome through their ability to recognize
N-acetyl lysine (KAc) post-translational modifications on
histone tails. These interactions have been implicated in
various disease states and, consequently, disruption of BET−
KAc binding has emerged as an attractive therapeutic strategy
with a number of small molecule inhibitors now under
investigation in the clinic. However, until the utility of these
advanced candidates is fully assessed by these trials, there
remains scope for the discovery of inhibitors from new
chemotypes with alternative physicochemical, pharmacoki-
netic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole
compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library
technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-
based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is
projected to have a low human efficacious dose.
investigation.5 Most of these inhibitors bind to each of the
four BET isoforms and their tandem bromodomain modules
INTRODUCTION
■
Protein transcription is a tightly regulated process and is
with similar affinity, that is, pan-inhibition, but reports of
controlled, in part, by proteins which “read” post-translational
modifications (PTMs) on flexible histone tails.1 One important
molecules selective for either the first [(N-terminal)
bromodomain, BD1] or second [(C-terminal) bromodomain,
class of the reader module, known as bromodomains,
recognizes N-acetyl lysine (KAc) PTMs, and dysregulation of
BD2] set of bromodomains have emerged,6−9 as well as
isoform-selective inhibitors10 and compounds selective for one
out of the eight BET bromodomains.11 Furthermore, the BET
family has been targeted using contemporary approaches such
as bivalent inhibition of the tandem bromodomain mod-
ules,12−14 degradation via the proteasome using proteolysis
targeting chimera technology,15−19 and through covalent
inhibition.20
their function can lead to pathological conditions.2 Of the 46
bromodomain-containing proteins (BCPs) identified,3 the
bromodomain and extraterminal domain (BET) family
(BRD2, BRD3, BRD4, and BRDT) have been the most
extensively studied, with links established to a diverse range of
disease states such as cancer, inflammation, and viral
infection.4 Because of these associations, abrogation of
BET−KAc interactions is a burgeoning field of research with
an increasing number of small molecule inhibitors being
described, several of which are currently under clinical
Received: October 8, 2019
© XXXX American Chemical Society
A
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A key goal in the development of BET inhibitors toward
medicines has been to establish activity in preclinical species,
and success in this area was demonstrated early in the field. For
example, a seminal study with I-BET762 1 (Figure 1)
both favorable pharmacokinetic parameters and high potency
for the target to achieve a low predicted human efficacious
dose and minimize the risk of idiosyncratic toxicity.
Accordingly, we herein report the development of a potent
and soluble dimethylpyridone benzimidazole BET inhibitor
exhibiting PK profiles which, after single species scaling based
on the allometric principles, correspond to low predicted
human doses of <30 mg once every day (QD). Hybridization
of a dimethylphenol benzimidazole series identified using
encoded library technology (ELT) with a methyl pyridone
series discovered by fragment screening resulted in an
improved dimethylpyridone benzimidazole scaffold. Subse-
quent optimization of off-target bromodomain selectivity was
carried out alongside frequent PKPD modeling to deliver an
inhibitor which fully engaged the BET mechanism and
demonstrated immunomodulatory effects in vivo.
RESULTS AND DISCUSSION
■
Our overall goal was to discover an orally bioavailable BET
inhibitor that was structurally distinct from our existing clinical
candidates.22,47 We set approximate potency criteria of <50
nM in a BET biochemical fluorescence resonance energy
transfer (FRET) assay and <100 nM in an LPS-stimulated
human whole blood (WB) assay measuring inhibition of the
proinflammatory cytokines monocyte chemoattractant protein
1 (MCP-1) and interleukin 6 (IL-6), a phenotype of BET
inhibition.20 We also required 100-fold selectivity over other
BCPs and over representative targets from other target classes.
To minimize the risk of compound-based attrition, we aimed
for a property forecast index (PFI [ChromLogD7.4 + number of
aromatic rings])48 <6, solubility in FaSSIF >100 μg/mL, and a
predicted human daily dose <100 mg. This was in accordance
with our oral candidate quality criteria,49 and these parameters
guided the optimization process.
To identify a novel BET inhibitor chemotype, we embarked
on a screening campaign using ELT which has proven to be an
important lead generation platform for various therapeutic
targets.50−55 A 6His-BRD4 (1−477) tandem bromodomain
construct was screened against a panel of DNA-encoded small-
molecule libraries using affinity-based selection technology,
and a hit series was identified from a three-cycle benzimidazole
core library (Figure 2). This specific library was constructed
using a split-and-pool strategy with three cycles of building
blocks (BBs) to provide a total warhead diversity of 117
million compounds as previously reported.53−55 Nucleophilic
aromatic substitution of DNA-appended 4-fluoro-3-nitro-
benzoate with 65 monoprotected diamines at cycle 1 was
followed by nitro reduction to afford 65 benzenediamines
(Figure 2a). These diamines were condensed with 922
aldehydes at cycle 2 to form benzimidazole ring systems.
After amine deprotection, the library was reacted with 1960
amine capping groups (carboxylic acids, isocyanates, sulfonyl
chlorides, and heterocyclic halides) to afford the final library.
DNA tags were added after each cycle of reaction to
unambiguously encode each warhead in the library. Inter-
pretation of the affinity selection from this library was viewed
in a cubic scatter plot (Figure 2b), where each axis corresponds
to a BB cycle. Examination of these data revealed enrichment
of a specific BB1 (piperidin-4-ylmethanamine, 5) and BB2 (4-
hydroxy-3,5-dimethylbenzaldehyde, 6) combination which
corresponded to 1,2,5-substituted benzimidazole 7. The cycle
3 amine capping groups were predominately carboxylic acids of
diverse structure, and the appearance of a highly populated
Figure 1. (a) Domain architecture of BET highlighting the eight
bromodomains. (b) Early generation BET inhibitors with in vivo
capability.
demonstrated its ability to protect mice against death in
models of endotoxic shock and sepsis,21 and it was
subsequently shown to inhibit ear swelling in a rat model of
delayed-type hypersensitivity.22 The related BET inhibitor,
JQ1 2, was first shown to reduce the tumor size and promote
survival in mouse xenograft models of nuclear protein in testis
midline carcinoma,23 and has since been evaluated in a range
of animal models including male contraception,24 bleomycin-
induced lung fibrosis,25,26 and cardiac hypotrophy.27 Another
molecule first published at the start of the last decade, RVX-
208 3, has been shown to increase apolipoprotein A-1 and
high-density lipoprotein cholesterol in monkeys28 and reduce
atherosclerosis in hyperlipidemic apolipoprotein E-deficient
mice.29 The early generation BET inhibitor, I-BET151 4,
promoted survival in mouse models of leukemia30 and,
similarly to I-BET762 1, suppressed cytokine production and
protected against death in acute inflammatory mouse
models.31,32 Data from in vivo studies with newer inhibitors
are emerging,33−46 but such reports often disclose response
data without the corresponding drug concentrations, which
prevents full understanding of pharmacokinetic/pharmacody-
namic (PKPD) effects and can hinder modeling predictions for
humans. Furthermore, some inhibitors have been reported to
display solubility-limited absorption following oral dosing32
and short half-lives22,23,39 and are at risk of not engaging the
mechanism in humans if poor systemic exposure translates. For
these reasons, there remains scope for the development of BET
inhibitors with sufficient exposure in multiple preclinical test
species to provide confidence that the mechanism can be
engaged in humans. Furthermore, inhibitors need to possess
B
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Figure 2. (a) Scheme of the hit DNA-encoded benzimidazole library where BB1, BB2, and BB3 refer to cycle 1, 2, and 3 BBs, respectively. (b)
Result of the BRD4 screen of the benzimidazole library. Compounds on the highlighted horizontal line (light blue) in the cubic scatter plot shared
the common BB1−BB2 combination (5 and 6, respectively) with BB3 as a variable component resulting in the generic hit structure 7. The sphere
size is proportional to the DNA tag copy number. Library members with less than 5 copies were removed to simplify visualization.
(light blue) line along the x-axis indicated no preference for
any particular carboxylic acid, suggesting that BB3 contributed
little to the binding.
carried out in one pot, whereby the ortho-nitroaniline was
reacted with 4-hydroxy-3,5-dimethylbenzaldehyde in the
presence of sodium dithionite in a nitro reduction−
aromatization sequence according to the method of Yang et
al.56 Subsequent N-Boc removal provided amine 8 which was
acylated to provide the target amides 9 and 10. The activities
of these compounds were measured in a historical fluorescence
polarization (FP) assay format and/or a routine FRET assay
format using a mutant BRD4 construct which determined
activity at BD1 and was used as a surrogate for activities at all
BET bromodomains (Table 1). Lipophilicities were measured
using a chromatographic method at pH = 7.4 (Chrom-
LogD7.4),48 and the corresponding PFI values were calculated.
Piperidine 8 was weakly active at BRD4 but potency was
increased by capping as the benzamide 9 and acetamide 10,
indicating a basic center was not optimal for binding to the
target at this position in the molecule. While acetamide 10 was
the slightly less potent amide, it was less lipophilic than
benzamide 9 leading to a more desirable PFI and higher LLE.
Therefore, exploration of alternative benzimidazole N1
substituents was generally conducted with acetamides in
place. First, however, we were interested in assessing the
importance of the benzimidazole 5-position amide toward
binding affinity, given its role as the linking functionality to
DNA during ELT hit identification. Accordingly, we prepared
benzimidazole 15a lacking the 5-amide following Scheme 2
and, after biochemical testing, found only a slight lowering in
activity versus its amide-substituted congener 10 (BRD4 FP
pIC50 = 6.3 and 6.6, respectively). Therefore, further
exploration of SAR at the benzimidazole N1-position was
conducted without the 5-amide in place (compounds 15b−g,
Scheme 2); BRD4 FRET assay results are shown in Table 2.
Having identified a hit series, we prepared several 1,2,5-
substituted benzimidazoles without DNA tags as exemplars of
the hit BB1−BB2 combination 7 in a similar manner to that
outlined in Figure 2a (Scheme 1). The synthetic sequence
commenced with reaction of 4-fluoro-N-methyl-3-nitrobenza-
mide with tert-butyl 4-(aminomethyl)piperidine-1-carboxylate
(5) leading to the nucleophilic aromatic substitution product
which was used without purification in the formation of the
benzimidazole ring. Construction of the heterocycle was
Scheme 1. Synthesis of Dimethylphenol Benzimidazole
a
Analogues 8−10
a
Reagents and conditions: (a) (i) tert-butyl 4-(aminomethyl)-
piperidine-1-carboxylate (5), DIPEA, EtOH, 80 °C; (ii) 4-hydroxy-
3,5-dimethylbenzaldehyde, Na2S2O4, EtOH/H2O (4:1), 80 °C; (iii)
TFA, DCM, rt; (b) PhCO2H, HATU, DIPEA, DMF, rt; (c) (i) Ac2O,
pyr, THF/MeCN; (ii) 2 M LiOH, THF/MeOH (4:1), rt.
C
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Table 1. Data for Dimethylphenol Benzimidazole N1 Analogues 8−10
a
BRD4 pIC50
b
b c
,
compound
R
FP
FRET
ChromLogD7.4
PFI
LE
LLE
8
9
10
H
NT
6.9
6.6
5.7
6.7
NT
0.8
3.2
2.0
3.8
7.2
5.0
/0.27
/2.1
2.5/2.3
4.0/
PhC(O)
CH3(O)
0.26/0.25
0.28/
a
b
c
Expressed as the mean from at least two test occasions. NT = Not tested. Calculated from FP/FRET pIC50 values. Defined as BRD4 pIC50
−
clog P.
a
Scheme 2. Synthesis of Dimethylphenol Benzimidazole N1 Analogues 15a−g
a
Reagents and conditions: (a) RNH2, K2CO3, MeCN, 80−110 °C; (b) H2, Pd/C, DMF, 30 °C; (c) 4-hydroxy-3,5-dimethylbenzaldehyde,
Na2S2O5, DMF, 100 °C; (d) 4 M HCl in MeOH, rt−50 °C; (e) AcOH or 2-hydroxyacetic acid, BOP, NEt3, DCM, rt.
BRD4 activity for 4-piperidine acetamide 15b was threefold
less than that for 4-methylene piperidine acetamide 15a and
highlighted the importance of a linking methylene between the
pendant heterocycle and the benzimidazole core. The size of
the heterocycle also affected activity, underscored by the lower
potencies for azetidine 15c and racemic pyrrolidine 15d
compared to piperidine 15a. Transposition of the amide in
piperidine acetamide 15a to an exocyclic position gave the
cyclohexyl acetamides 15e and 15f, and activity was
maintained with the latter cis-isomer. Finally, it was
demonstrated that the acetamide 15a could be substituted
with a hydroxyl group to give the more polar hydroxy
acetamide 15g without lowering BRD4 activity.
During these investigations, we pursued efforts to obtain an
X-ray crystal structure of acetamide 10. Gratifyingly,
cocrystallization with BRD4 BD1 resulted in a 1.5 Å structure
and revealed the dimethylphenol moiety positioned in the KAc
binding site (Figure 3).
The 2,6-dimethylphenol group replicated the interactions of
KAc3 and bound in the same manner as other 2,6-
dimethylphenol-containing BET ligands.57 Specifically, the
hydroxyl accepted a hydrogen bond from Asn140 and donated
a hydrogen bond via water to Tyr97, while an ortho-methyl
substituent occupied a lipophilic pocket adjacent to Phe83.
The phenyl ring of the benzimidazole bicyclic system was
positioned between Leu92 and Trp81 (a region known as the
ZA channel) and formed an edge-to-face interaction with the
latter residue. The benzimidazole N1 position provided an
appropriate vector for the 4-methylene piperidine to bind to
the lipophilic region flanking the Trp81-Pro82-Phe83 triad,
commonly referred to as the WPF shelf, and occupying this
part of the binding site has generally been associated with
increased potency.58 The relatively small Ile146 residue in
BRD4 BD1, located near the start of the C-helix, permitted
access to the WPF shelf but larger residues at this position in
other BCPs preclude access, and thus, binding to the WPF
shelf in BRD4 BD1 was expected to impart selectivity over
certain BCPs.58 The acetamide was directed out of the WPF
shelf toward the solvent, and this was consistent with SARs
demonstrating that alterations to the amide did not profoundly
affect potency. The methylamide substituent at the 5-position
of the benzimidazole was directed out of the ZA channel
toward the solvent, and this positioning was consistent with its
role as the linker to DNA during hit identification. A final
observation was that the benzimidazole N3 atom was
sufficiently close (2.7 Å) to accept a hydrogen bond from an
adjacent water molecule, which in turn formed contacts to
D
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With the binding mode and opportunity for structure-based
design established, we were keen to investigate this series
further and identify any issues requiring optimization. Our
primary concern at this early stage was the reliance on the 2,6-
dimethylphenol moiety as the KAc mimetic. Given the general
requirement for BET inhibitors to possess a KAc mimetic,5 the
2,6-dimethylphenol was deemed integral to achieve high
potency but represented a potential liability because of the
propensity of phenols to be rapidly metabolized by phase II
enzymes. Therefore, dimethylphenol 10 was assessed in a
mouse PK experiment where clearance from the blood (346
mL/min/kg) was found to be greater than liver blood flow,
and a short half-life (0.37 h) was observed after iv dosing (see
the Supporting Information). Based on these results, we
commenced investigations to replace the 2,6-dimethylphenol
KAc mimetic.
Table 2. BRD4 Activities and PFI Values of Dimethylphenol
Benzimidazole N1 Analogues 15a−g
To find suitable alternative KAc-binders and integrate
additional information for optimal lead generation,59 we
turned to our extensive collection of bromodomain fragment
data. We have previously described our first screen of a
fragment library against the BET family bromodomains, which
generated multiple chemically diverse low-molecular-weight
starting points that bind in the KAc site.60 These are ligand-
efficient starting points, and we have shown that they can be
optimized for BET bromodomains using structure-based
design to build in the extra pharmacophoric components
needed for high-affinity binding.61 In addition, we have found
the pool of multiple bromodomain/fragment crystal structures
to be a valuable source of ideas for isosteric replacements and
hybridization during optimization of leads from other sources
including high-throughput screening and ELT.
One of the fragments emerging from this work was the low-
affinity ligand-efficient N-methyl pyridone-containing com-
pound 16 (BRD4 FRET pIC50 = 4.9, LE = 0.42) (Figure 4a).
A 1.9 Å X-ray crystal structure of this fragment in BRD2 BD1
showed the pyridone group binding in the KAc-site, with its
carbonyl group forming a direct hydrogen bond interaction
with Asn156 and through the water network to Tyr113 (Figure
4b). The methoxyphenyl group partially occupied the ZA
channel without completely filling it.
a
Expressed as the mean from at least three test occasions.
A substructure search for related N-methyl pyridone
analogues in the GSK compound collection identified the
imidazole 17 (BRD4 FRET pIC50 = 5.2, LE = 0.36). A 2.3 Å
X-ray crystal structure of this compound was also obtained in
BRD2 BD1 (Figure 4c) and confirmed that the interactions of
the N-methylpyridone are the same as those made by fragment
16. The imidazole pointed toward the ZA channel with one of
its nitrogens forming a hydrogen bond to water. Although the
fluorophenyl ring did not occupy the WPF shelf, this
substituent position on the imidazole offered a vector to
access the shelf and potentially gain potency.
Figure 4d shows the superimposed crystal structures of
pyridone 17 in BRD2 BD1 and the phenol 10 in BRD4 BD1.
Unusually, in bromodomain-inhibitor complexes, the phenolic
OH group of 10 probably acts as a hydrogen bond donor
toward the W1 water. This contrasts with the carbonyl of
pyridone 17 which can only act as a more typical hydrogen
bond acceptor to the W1 water. To accommodate this
difference would require a rotation of the W1 water and a
change of some of its interactions with the conserved tyrosine
or the other conserved water molecules (Figure 4), but it is not
possible to see the details from the electron density. We
speculate that the requirement for an uncommon water
Figure 3. X-ray crystal structure of acetamide 10 (carbon = green) in
BRD4 BD1 (carbon = gray, PDB 6TPX). Water molecules at the base
of the binding pocket are shown as red spheres, and selected hydrogen
bonds are shown as yellow dashed lines.
other water molecules and Pro82 and was therefore deemed an
important interaction.
E
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Table 3. BRD4 Activities and PFI Values of Benzimidazole
2-Position Analogues 19a−d
a
b
Expressed as the mean from at least two test occasions. A pIC50
value of <4.8 was determined on one test occasion out of four and was
excluded from the reported mean value.
Figure 4. (a) Identification of pyridones as KAc-mimetics from a
fragment-based screen.59 (b) X-ray crystal structure of N-methyl
pyridone fragment 16 (carbon = orange) in BRD2 BD1 (PDB
6TQ1). (c) X-ray crystal structure of N-methyl pyridone imidazole 17
(carbon = cyan) in BRD2 BD1 (PDB 6TQ2). (d) Superposition of X-
ray crystal structures of N-methyl pyridone imidazole 17 (carbon =
cyan) in BRD2 BD1 (PDB 6TQ2) and 2,6-dimethylphenol 10
(carbon = green) in BRD4 BD1 (PDB 6TPX).
methyl substituents was established by testing mono C3-
methyl 19b and mono N1-methyl 19c and both lowered
activity relative to dimethyl 19a but to different extents.
Removal of both methyl groups to give NH pyridone 19d
resulted in the loss of measurable activity.
Notwithstanding the encouraging biochemical and physi-
ochemical data for 1,3-dimethylpyridone 19a, the BRD4
activity and MCP-1 WB potency (pIC50 = 5.8) were still
below our target levels. We therefore sought to replace the
benzimidazole N1-substituent with a more potent group. SARs
established during off-DNA ELT chemistry (data not shown)
indicated that lipophilic substituents were preferred at this
position, and this was consistent with binding to the nonpolar
residues which comprise the WPF shelf (see Figure 3). The
reduction in lipophilicity afforded by replacing the dimethyl-
phenol KAc mimetic with the dimethylpyridone allowed
investigation of a substituent lacking a polar amide group
while maintaining the overall physicochemical properties in a
orientation to bind the phenols may explain their lower ligand
efficiency relative to the pyridone. Regardless of this, the close
overlay between the crystal structures of these series suggested
that we should attempt a hybridization approach. It is worth
noting that other pyridone-containing BET inhibitors have
been reported since this work was carried out.62−66
Preparation of hydrid 1,3-dimethylpyridone 19a and close
analogues was carried out according to Scheme 3, and
biological results are shown in Table 3. Pleasingly, BRD4
activity for 1,3-dimethylpyridone 19a was higher than that for
2,6-dimethylphenol 15g, and lipophilicity was lowered by ∼10-
fold which resulted in a higher LLE. The requirement for two
a
Scheme 3. Synthesis of Benzimidazole 2-Position Analogues 19a−d
a
Reagents and conditions: for compounds 18a, 18b, and 18d: (a) (i) RCHO, Na2S2O5, DMF, 100 °C; (ii) HCl in MeOH, rt; for compound 18c:
(b) RCO2H, PPA, 180 °C; (c) 2-hydroxyacetic acid, EDC·HCl, HOBt, DIPEA, DCM, rt.
F
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desirable space. This led us to prepare 4-methylene
tetrahydropyran (THP) 20a (Scheme 4) which exhibited
contributed to effective suppression of the proinflammatory
cytokines IL-6 and MCP-1 in LPS-stimulated human WB and
high solubility in FaSSIF (determined using a crystalline solid
form).
Scheme 4. Synthesis of Dimethylpyridone Benzimidazole
a
Having identified an attractive lead compound, we carried
out more comprehensive profiling to identify and understand
any issues to guide the medicinal chemistry strategy.
Accordingly, we screened 4-methylene THP 20a against a
panel of eight representative BCPs (see the Supporting
Information) and found that it bound to bromodomain
adjacent to zinc finger domain 2A (BAZ2A) with notable
affinity (Table 4). BAZ2A has been identified as the large
subunit of the nucleolar remodeling complex and plays a role
in chromosome stability, and it is known to silence some rRNA
genes and can delay proliferative growth when knocked down
in cells.67 It was unknown how this profile could translate to
humans, so we set the goal to overcome this off-target activity
during lead optimization. We also conducted PK profiling on
4-methylene THP 20a to benchmark the pyridone benzimi-
dazole series. Initially, compound stability in microsome and
hepatocyte preparations was assessed and highlighted differ-
ences between species with rats displaying higher turnover
than both humans and dogs (Table 5). Comparison of these
data with observed in vivo clearance following iv admin-
istration demonstrated a good correlation. The high clearance
observed in rats required improvement to ensure adequate
systemic exposure following oral administration for preliminary
toxicology studies.
N1 Analogues 20a−i
a
Reagents and conditions: (a) amine, DIPEA, DMSO or NMP or 2-
MeTHF, microwave, 110−125 °C; (b) (i) 1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carbaldehyde, Na2S2O4, EtOH/H2O (2:1), 100
°C; (ii) for compound 20e: TFA, DCM, rt; (c) (i) H2, Pd/C, EtOH,
rt; (ii) Oxone, DMF/H2O (30:1), rt.
At this stage, the human efficacious dose of 4-methylene
THP 20a was predicted using the single species scaling
approach68,69 (correcting for differences in the fraction
unbound) in conjunction with the MCP-1 WB activity. A
target coverage of IC50 over a 24 h dosing period was selected
at this stage of our investigations for sufficient engagement of
the mechanism, and the predicted once-daily human doses
based on this were >2 and 1.4 g from rats and dogs,
respectively (see the Supporting Information for details and
assumptions). These predicted high human doses were a result
of the moderate WB MCP-1 activity combined with a
predicted short half-life. With an aim to have a dose <100
mg, these contributing factors became a focus for our future
optimization efforts.
During profiling lead benzimidazole 20a, we obtained a 1.8
Å resolution X-ray crystal structure in BRD4 BD1, and this
revealed an identical binding mode to that of hit benzimidazole
10 with direct overlay of the 1,3-dimethylpyridone and 2,6-
dimethylphenol moieties (Figure 5a). This structure also
provided a basis to interrogate the observed binding to BAZ2A
through superposition of an apo structure of BAZ2A70 (Figure
5b). This showed several differences most notably in the C-
helices where Met149 in BRD4 BD1 is replaced by Ala1882 in
<100 nM activity at BRD4 and possessed favorable
physicochemical properties (Table 4). These characteristics
Table 4. Structure of 4-Methylene THP 20a and Initial
Profile
a
BRD4 FRET pIC50
7.1
6.7/6.6
6.5
a
WB pIC50 IL-6/MCP-1
BAZ2A FRET pIC50
b
ChromLogD7.4/PFI
LE/LLE
2.7/5.7
0.39/4.8
FaSSIF (μg/mL)
>1000
a
b
Expressed as the mean from at least four test occasions. Expressed
as the mean from two test occasions.
Table 5. In Vitro and in Vivo PK Data of 4-Methylene THP 20a
IVC (mL/min/g)
species
Mics
Heps
fub
CLb (mL/min/kg) CLu (mL/min/kg) Vdss (L/kg)
T1/2, iv (h) F (%) human predicted QD dose (mg)
a
rat
3.2
<0.37
0.83
17
<1.3
<0.87
0.55
0.47
0.40
82
8
149
17
1.4
1.4
0.2
2.3
>2000
1400
b c
,
dog
human
97
a
b
iv dose (1.0 mg/kg): 1 h infusion in DMSO (2%, v/v) and Kleptose HPB (10%, w/v) in saline (0.9% w/v). iv dose (0.5 mg/kg): 1 h infusion in
c
DMSO (2%, v/v) and Kleptose HPB (10%, w/v) in saline (0.9% w/v). po dose (1.0 mg/kg): a suspension of solid of unknown form in 1% (w/v)
methylcellulose (400 cps) (aq).
G
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Figure 5. (a) Superposition of X-ray crystal structures of 1,3-dimethylpyridone 20a (carbon = cyan, PDB 6TPY) and 2,6-dimethylphenol 10 in
BRD4 BD1 (carbon = green, PDB 6TPX). A surface was applied to the protein of PDB 6TPX. (b) View at the base of the binding pocket from
superposition of 1,3-dimethylpyridone 20a (carbon, water molecules [spheres], and selected hydrogen bonds [dashed lines] = cyan PDB 6TPY)
and 2,6-dimethylphenol 10 (carbon, water molecules [spheres], and selected hydrogen bonds [dashed lines] = green, PDB 6TPX) X-ray crystal
structures in BRD4 BD1. A surface was applied to the protein of PDB 6TPX. (c) Superposition of the X-ray crystal structure of 1,3-
dimethylpyridone 20a in BRD4 BD1 (carbon = cyan, PDB 6TPY) with an apo structure of BAZ2A70 (carbon = orange, PDB 4LZ2). A wireframe
surface has been applied to Trp1816 of BAZ2A.
BAZ2A. The consequence of this difference is the collapse of
the WPF motif (consisting of Trp1816-Pro1817-Phe1818) and
a closed WPF-shelf in BAZ2A, whereas this region is fully open
and accessible in the BET family. It was clear that this
superposition did not provide an explanation for the observed
BAZ2A activity for lead compound 20a as the THP moiety
would sterically clash with Trp1816 and preclude binding to
this BCP. Another effect of the collapsed WPF motif in BAZ2A
was the enlargement of the ZA channel relative to that in
BRD4 BD1, and this was increased further by deletions in the
ZA loop indicating that Leu1826 was unable to form a narrow
junction equivalent to that caused by Leu92 in BRD4 BD1. We
speculated that the enlarged ZA channel in BAZ2A permitted
distortion of the dimethylphenol-benzimidazole dihedral angle
from twisted (42°) in BRD4 BD1 to a more coplanar
conformation in BAZ2A and that branching off the methylene
group attached to the benzimidazole N1 position would
restrict this conformational freedom, thereby increasing
selectivity over BAZ2A.
Using this structural insight, we investigated branching on
the methylene group and other modifications to the 4-
methylene THP and counter-screened for their BAZ2A
activities (Scheme 4 and Table 6). An additional consideration
during this exploration was to limit significant increases in
lipophilicity.
Incorporation of a methyl branch on the methylene gave the
enantiomeric pair 20b and 20b′ which was obtained upon
chiral chromatographic separation of the racemic mixture. In
terms of BRD4 FRET activity, a clear preference for
stereoisomer 20b′ was observed, but BAZ2A activity was not
significantly affected when compared to unbranched methylene
20a. However, further extension of the branching substituent
did reduce BAZ2A activity with ethyl enantiomers 20c and
20c′ exhibiting preferential binding to BRD4 by >125- and 40-
fold, respectively. Strikingly, the 3-methylene THP enan-
tiomers 20d and 20d′ reduced BAZ2A activity by several
orders of magnitude compared to 4-methylene THP 20a with
the latter enantiomer displaying particularly high BRD4
activity resulting in ∼3000-fold selectivity. Other simple
modifications to the 4-methylene THP group, such as moving
the ether oxygen to an exocyclic position to give 4-methylene
cyclohexylalcohol 20e and homologating the methylene to give
ethylene 20f, lowered BAZ2A activity while maintaining BRD4
potency. The spirocyclic oxetane 20g, a motif used as a
replacement for 6-membered cyclic systems,71,72 also increased
selectivity over BAZ2A and, additionally, lowered microsomal
clearance (rat and human = 0.99 and <0.40 mL/min/g,
respectively) while remaining liponeutral compared to THP
20a. Acyclic substituents were also explored with racemic
propyl methoxy 20h demonstrating >100-fold selectivity over
BAZ2A but only moderate BRD4 activity. Achiral dimethox-
ypropyl 20i maintained a high level of BRD4 activity and was
inactive at BAZ2A. Taken together, these data revealed the 4-
methylene THP group to be a privileged motif for binding to
BAZ2A and only minor modifications were required to
dampen activity.
We further profiled dimethoxypropyl 20i and found that WB
potencies (pIC50 = 6.6 and 7.0 for IL-6 and MCP-1 analytes,
respectively) were commensurate with high BRD4 FRET
activity. In addition, high solubility was determined in FaSSIF
media (>1000 μg/mL) from a crystalline solid form. We also
assessed the compound for stability in microsomes and
hepatocytes and found that it was more stable in both rat
and human preparations versus 4-methylene THP 20a (Table
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Table 6. BRD4 and BAZ2A Activities and ChromLogD7.4 Values of Benzimidazole N1 Analogues 20a−i
a
b
c
Expressed as the mean from at least two test occasions. Calculated as BRD4 FRET pIC50 − BAZ2A pIC50. pIC50 values of <4.0 were determined
on two test occasions out of four and were excluded from the reported mean value.
7). This improvement was noteworthy, given the slight
increase in lipophilicity incurred upon replacement of the 4-
methylene THP with the dimethoxypropyl and, therefore,
represented an alternative strategy for improving the stability
of this cyclic ether compared to reducing the ring size.73
A
subsequent in vivo rat PK study revealed moderate clearance
and a longer half-life relative to 4-methylene THP 20a. The
clearance, and unbound clearance, of dimethoxypropyl 20i in
I
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Table 7. In Vitro and in Vivo PK Data of Dimethoxypropyl 20i
IVC (mL/min/g)
species
Mics
Heps
fub
CLb (mL/min/kg) CLu (mL/min/kg) Vdss (L/kg)
T
1/2, iv (h) F (%) human predicted QD dose (mg)
ab
,
rat
<0.46
<0.37
<0.40
1.1
<1.3
<0.87
0.59
0.57
0.37
37
5
63
9
2.1
0.8
3.2
1.7
46
46
1600
470
c d
,
dog
human
a
b
iv dose (1.5 mg/kg): 1 h infusion in DMSO (2%, v/v) and Kleptose HPB (10%, w/v) in saline (0.9% w/v). po dose (3 mg/kg): a suspension of
c
crystalline solid in 1% (w/v) methylcellulose (400 cps) (aq). iv dose (0.5 mg/kg): 1 h infusion in DMSO (2%, v/v) and Kleptose HPB (10%, w/
v) in saline (0.9% w/v). po dose (1.0 mg/kg): a suspension of crystalline solid in 1% (w/v) methylcellulose (400 cps) (aq).
d
a
Scheme 5. Synthesis of Dimethylpyridone Benzimidazole 5- and 6-Position Analogues 22a−p
a
Reagents and conditions: for compounds 21a and 21b: (a) 4-substituted 1-fluoro-2-nitrobenzene or 4-substituted 4-bromo-2-fluoro-1-
nitrobenzene, (tetrahydro-2H-pyran-4-yl)methanamine, DIPEA, 2-MeTHF, 80 °C; for compounds 22a and 22b: (b) 1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carbaldehyde, Na2S2O4, EtOH/H2O (2.5:1), 90 or 80 °C; (c) HN(CH3)2, Pd2(dba)3 (2 mol %), JohnPhos (13 mol %),
NaOtBu, toluene or 1,4-dioxane, microwave, 90 °C; for compounds 22e−22p: (d) (i) substituted 1-fluoro-2-nitrobenzene, (tetrahydro-2H-pyran-
4-yl)methanamine, DIPEA, 2-MeTHF, microwave, 110 °C; (ii) 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde, Na2S2O4, EtOH/H2O
(2:1), 100 °C.
Figure 6. Plots of BRD4 FRET mean activities vs sigma para values for substituents at the 5-position (left) and 6-position (right).
dogs was slightly lower than that observed with 4-methylene
THP 20a, but a lower Vdss was determined resulting in a
shorter T1/2. These PK data, combined with the WB MCP-1
activity, were used to calculate the predicted once-daily human
doses of 1.6 g and 470 mg from rat and dog data, respectively.
Although the predicted doses were lower than those for 4-
methylene THP 20a, a further lowering of the projected dose
was required to meet our target criterion of <100 mg.
In parallel with investigations at the benzimidazole N1-
position, we pursued explorations at the 5- and 6-positions
aiming to lower the predicted human dose versus lead
compound 20a based on a dual approach. First, an increase
in potency was predicted by substituting these positions based
on the crystal structure of 4-methylene THP 20a in BRD4
BD1 (see Figure 5a). Here, the phenyl ring of the
benzimidazole formed an edge-to-face interaction with
Trp81, and we therefore hypothesized that increasing electron
density in the ring would improve this interaction and lead to
increased activity; it was also noted that the interaction to
water through the benzimidazole N3 nitrogen could be
modified through such electronic effects. Second, reports of
unsubstituted benzimidazole rings being metabolically vulner-
able74,75 led us to block the 5- and 6-positions with appropriate
substituents to lower the clearance and increase systemic
exposure and the half-life.
We sought to test the first hypothesis by preparing analogues
where the 5- and 6-positions were substituted with both
electron-donating and electron-withdrawing groups. Synthesis
J
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of these compounds was carried out using the established
SNAr-cyclization sequence, which, in the case of the dimethyl-
amine target compounds 22c and 22d, required subsequent
C−N cross-coupling of the Br-substituted benzimidazoles 22a
and 22b, respectively (Scheme 5).
BRD4 FRET data were obtained for these compounds, and
the activities were plotted against sigma para values76 (Figure
6). A general correlation for increased electron density with
increased potency was observed for both the 5- and 6-positions
(r2 values of 0.52 and 0.48, respectively) which supported our
hypothesis of improving the benzimidazole-Trp81 edge-to-face
and/or the N3-water interaction(s).
As outlined previously, we were also interested to examine
the effects upon metabolic stability by incorporating these
substituents on the benzimidazole ring. Toward this end,
potent dimethylamino 5-substituted benzimidazole 22c was
assessed in microsome preparations and levels of turnover were
below the lower limit of quantification in all species (see the
Supporting Information). This result was noteworthy, given
the similar lipophilicities for 5-dimethylamine 22c and
unsubstituted benzimidazole 20a (ChromLogD7.4 = 2.9 and
2.7, respectively), and corroborated our strategy of blocking
potential sites of metabolism (see Table 5 for microsome
clearance data).
Based on the encouraging activities for the dimethylamines
22c and 22d and the low microsome clearance for the former
5-substituted congener, we prepared further amines at both the
5- and 6-positions to determine the preferred site of
substitution for activity (Scheme 6).
5-position was preferred for activity in most cases (Figure 7).
This observation also held true for the WB activity where the
5-position substituents were more potent in 10 out of the 11
MMPs. Furthermore, most 5-substituents increased BRD4
FRET and MCP-1 WB activity relative to 5-dimethylamine
22c. The 5-morpholine 23e was particularly potent in both the
biochemical and MCP-1 WB assays (pIC50 = 8.0 and 7.9,
respectively) and warranted further profiling. As expected, this
4-methylene THP-containing compound exhibited BAZ2A
activity (pIC50 = 6.7) corresponding to only 20-fold selectivity
for BRD4. Nonetheless, we sought to confirm the earlier
observation that substitution at the 5-position improved
metabolic stability compared to unsubstituted benzimidazole
20a. Similar to 5-dimethylamine 22c, no detectable turnover of
5-morpholine 23e was measured in human microsomes (see
the Supporting Information). These latter data, combined with
the low measured lipophilicity (ChromLogD7.4 = 2.3) and high
FaSSIF solubility (>1000 μg/mL, from amorphous solid),
demonstrated the morpholine to be a favorable substituent at
the 5-position.
Having made improvements at both the benzimidazole N1-
and 5-positions independently, we sought to combine the
favored substituents into one molecule predicting that it would
display high BRD4 and low BAZ2A activity, while possessing
favorable PK and physicochemical properties for testing the
mechanism in vivo. Gratifyingly, our hypothesis was correct
and the profile of the resulting compound 24 (I-BET469)
(Table 8) is now discussed.
First, dimethylpyridone benzimidazole 24 displayed high
BRD4 FRET and WB activities which exceeded our target
levels. Activity at BRD4 in a cellular environment was also
assessed for this compound using NanoBRET technology.77
Here, HEK293 cells expressing a nanoluciferase-BRD4 fusion
protein were incubated with dimethylpyridone benzimidazole
24 and a bromosporine tracer. Displacement of the tracer was
monitored over 2 h resulting in a cellular pKd of 8.3, a value
greater than that determined for I-BET151 (pKd = 7.8). The
kinetics of binding were also measured and revealed a
marginally slower koff for dimethylpyridone benzimidazole 24
relative to I-BET151 (see the Supporting Information). With
regard to BCP selectivity, benzimidazole 24 was >5000-fold
selective over BAZ2A because of the inclusion of the N1-
dimethoxypropyl substituent. Screening in a panel of BCPs
revealed the overall high selectivity for the BET family (see the
Supporting Information); Kd values of 37 and 49 nM were
measured for CREBBP and EP300, respectively but were ≥25-
fold higher than that measured for BRD4 BD1 and did not
preclude further investigation of the compound.
We considered all matched molecular pairs (MMPs) of N-
substituents at both the 5- and 6-positions and found that the
Scheme 6. Synthesis of Dimethylpyridone Benzimidazole 5-
a
and 6-Position Amine Analogues 23a−23t
A 1.3 Å resolution X-ray crystal structure of dimethylpyr-
idone benzimidazole 24 in BRD4 BD1 was obtained (Figure
8) and showed the ligand bound in a comparable manner to
the previous 4-methylene THP benzimidazole 20a. Clear
differences with dimethoxypropyl benzimidazole 24, however,
were that the branched N1-substituent simultaneously
contacted the WPF-shelf and residues of the ZA loop
(Leu92 and Leu94) on the opposing side of the binding
pocket, and the morpholine 5-substituent was directed out of
the ZA channel toward the solvent.
The PFI value of dimethylpyridone benzimidazole 24 (5.6)
was within our desired criteria and therefore presented a low
risk toward compound-related attrition. At this stage, we were
keen to determine FaSSIF solubility from a crystalline form to
better understand its pharmaceutical properties and potential
a
Reagents and conditions: (a) (i) NHRR′, Pd2(dba)3 (2 mol %),
DavePhos (5 mol %), NaOtBu, toluene or 1,4-dioxane, 80 °C; for
compounds 23m and 23n: (ii) 4 M HCl in 1,4-dioxane, rt.
K
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Figure 7. Plots of MMP activities for 5-position (blue) and 6-position (green) substituents. BRD4 FRET data (left) and MCP-1 WB data (right).
Table 8. Profile of Dimethylpyridone Benzimidazole 24
for development. We also required further quantities of the
compound for in vivo studies, so synthesis of the crystalline
material was carried out on a >220 mmol scale (Scheme 7).
Nucleophilic substitution of 4-bromo-1-fluoro-2-nitroben-
zene with commercially available 1,3-dimethoxypropan-2-
amine afforded ortho-nitroaniline 25 and was followed by
ring formation to give benzimidazole 26. Palladium-catalyzed
C−N cross-coupling of bromobenzimidazole 26 with morpho-
line followed by recrystallization from EtOAc delivered 50 g of
the amination product 24. The solid state was confirmed as
crystalline via X-ray powder diffraction (see the Supporting
Information) and subsequent solubility measurements using
this material revealed solubility >1000 μg/mL in FaSSIF and
>820 μg/mL in other physiological media and aqueous buffers
(see the Supporting Information). With no detectable turnover
in rat, dog, and human microsomal and hepatocyte
preparations, the crystalline material was dosed in a crossover
rat PK study at 1, 3, and 30 mg/kg (Table 9). Following oral
administration, the dose-normalized AUC0−∞ values for each
dose group were equivalent (53 and 52 min·kg/L for the 3 and
30 mg groups, respectively), indicating a linear dose-exposure
relationship and because of the low clearance and high oral
absorption, oral bioavailabilities were high (ca. 94% following
administration at 3 and 30 mg/kg). Additionally, PK
parameters were determined in the dog and demonstrated
low clearance and high oral bioavailability. Single species
scaling of the rat and dog PK parameters predicted human
doses within our target of <100 mg (27 and 8 mg, respectively)
for 24 h coverage of the WB MCP-1 IC50. With the low
predicted human dose indicating low developability risk and
wider biological profiling demonstrating minimal off-target
activity (see the Supporting Information), we progressed
dimethylpyridone benzimidazole 24 into further studies.
a
BRD4 FRET pIC50
7.9
7.7/7.6
8.3
a
WB pIC50 IL-6/MCP-1
a
Cell BRET pKd
b
BAZ2A FRET pIC50
4.2
ChromLogD7.4/PFI
LE/LLE
2.6/5.6
0.35/6.4
a
b
Expressed as the mean from at least four test occasions. pIC50
values of <4.0 were determined on four test occasions out of 10 and
were excluded from the reported mean value.
Figure 8. X-ray crystal structure of dimethylpyridone benzimidazole
24 in BRD4 BD1 (carbon = magenta, PDB 6TPZ). Water molecules
at the base of the binding pocket are shown as red spheres, and
selected hydrogen bonds are shown as yellow dashed lines.
a
Scheme 7. Synthesis of Dimethylpyridone Benzimidazole 24
a
Reagents and conditions: (a) 1,3-dimethoxypropan-2-amine, K2CO3, MeCN, 80 °C, 97%; (b) 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-
carbaldehyde, Na2S2O4, EtOH/H2O (4:1), 90 °C, 56%; (c) morpholine, Pd2(dba)3 (2 mol %), DavePhos (5 mol %), NaOtBu, 2-MeTHF, 80 °C,
59%.
L
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Table 9. In Vitro and in Vivo PK Data of Dimethylpyridone Benzimidazole 24
IVC
(mL/min/g)
po dose
(mg/kg)
AUC0−∞
CLb
CLu
Vdss
T
1/2, iv
human predicted QD
dose (mg)
species
mouse
rat
Mics
Heps
fub
(ng·h/mL)
(mL/min/kg) (mL/min/kg) (L/kg)
(h)
F (%)
<0.48
<0.46
<1.0
<0.80
0.72
0.69
3.0
3.0
30.0
1.5
3618
2614
26973
12503
a b
,
17
2
24
4
2.3
0.9
3.8
94
93
101
27
8
c d
,
dog
<0.37
<0.40
<1.3
<0.87
0.50
0.72
5.3
human
a
b
iv dose (1.0 mg/kg): 1 h infusion in DMSO (2%, v/v) and Kleptose HPB (10%, w/v) in saline (0.9% w/v). po dose: a suspension of crystalline
c
solid in 1% (w/v) methylcellulose (400 cps) (aq). iv dose (0.5 mg/kg): 1 h infusion in DMSO (2%, v/v) and Kleptose HPB (10%, w/v) in saline
(0.9% w/v). po dose: a suspension of solid of unknown form in 1% (w/v) methylcellulose (400 cps) (aq).
d
Having established potent binding and functional activity in
vitro, we next wanted to demonstrate engagement of the BET
mechanism in vivo. An investigation by Tarakhovsky et al. was
the first to establish the link between BET inhibition and
inflammation.21 These researchers treated LPS-stimulated
bone marrow-derived macrophages with I-BET762 and
observed suppressed expression of proinflammatory genes
including IL-6. Epigenetic profiling provided further insight
into the mechanism with the promoter of IL-6 displaying
reduced BET recruitment in the presence of I-BET762.
Furthermore, experiments in vivo demonstrated that inhibition
of BET in a mouse model of endotoxic shock attenuated
release of IL-6. We therefore sought to use IL-6 as a biomarker
in LPS-challenged mice to establish the suitability of
dimethylpyridone benzimidazole 24 to engage the BET
mechanism in vivo. After confirming that adequate exposure
would be achieved following oral dosing to the male CD-1
mouse (Table 9), dimethylpyridone benzimidazole 24 was
administered orally at three different concentrations (1, 3, and
10 mg/kg) 30 min prior to LPS challenge and samples were
taken at regular intervals for PKPD analysis over the following
5 h. Immunoassay of mouse IL-6 revealed suppression of
cytokine levels in line with increasing unbound exposure over
the duration of the study (Figure 9).
antigen, and IgG1 levels were measured following QD or
once every other day (QOD) oral administration of
dimethylpyridone benzimidazole 24 for 14 days. Greater
inhibition of IgG1 levels relative to the vehicle on day 14 was
achieved in the 3 mg/kg QD versus the 0.3 mg/kg QD dose
group (20 and 65%, respectively) which was in accordance
with the higher average free drug concentrations (Cav) (Figure
10). Additionally, similar suppression of IgG1 levels compared
Figure 10. Mean %inhibition of IgG1 (relative to the vehicle) at day
14 vs free average drug concentration. Statistical differences in %
inhibition of IgG1 were determined as p < 0.01 (**).
to the vehicle was observed with the 3 mg/kg QOD regimen
versus QD (75 and 65%, respectively). The manifestation of
auto-antigen presentation to T cells, and consequent auto-
antibody production, fundamentally underlies auto-immune
disease. Therefore, the demonstration that BET inhibition can
attenuate antibody production in T cell-dependent immuniza-
tion may allow such an approach to be used for clinical benefit
in these conditions.
Taken together, these experiments demonstrate that
dimethylpyridone benzimidazole 24 possesses suitable proper-
ties to engage the BET mechanism in vivo and elicit potent
immunomodulatory effects. Moreover, the potency and oral
PK properties of this molecule provide the flexibility to model
different dosing regimens and levels of mechanism engagement
to refine human dose predictions for different disease
indications. Overall, the data presented here make this BET
inhibitor a potential candidate for assessment in humans.
Figure 9. IL-6 (red) and unbound dimethylpyridone benzimidazole
24 (blue) AUC concentrations in the acute LPS model of
inflammation. Statistical differences in %inhibition of IL-6 AUC
levels were determined as p <0.05 (*) and p <0.001 (***).
CONCLUSIONS
Having confirmed BET-mediated effects in an acute in vivo
model, we next investigated dimethylpyridone benzimidazole
24 in a chronic immune-mediated model more relevant to
human immune disease. The ability of BET inhibitors to
interfere with the immune response through dampened T cell
and B cell activation has previously been established.78,79 We
therefore utilized a T cell-dependent immunization model
wherein male CD-1 mice were challenged with the 2,4,6-
trinitrophenyl keyhole limpet hemocyanin (TNP-KLH)
■
We have described the discovery of a hit 3,5-dimethylphenol
benzimidazole series using ELT for an oral BET inhibitor
program. An X-ray crystal structure revealed the dimethylphe-
nol moiety to be the KAc mimetic. Hybridization of this with
an N-methylpyridone fragment-derived series led to increased
potency and lowered lipophilicity. A compound containing this
KAc mimetic was selected as a lead because of its high potency,
encouraging physicochemical properties, and excellent PK
M
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mm × 2.1 mm, 3 μm packing diameter). Elution was carried out at 35
°C with the mobile phases as (A) 0.1% formic acid in water and (B)
0.1% formic acid in acetonitrile using a gradient starting at 5% B,
increasing linearly to 100% B over 6 min, and remaining at 100% B for
2.5 min. The flow rate was 0.5 mL/min. The purity of all biologically
tested compounds was ≥95% as determined by LC−MS UV traces
except for compound 19b (93% purity). The specific LC−MS UV
method used for purity determination is noted for each compound
and is from any of the following: method A: Waters ZQ Acquity
UPLC system equipped with an CSH C18 column (50 mm × 2.1
mm, 1.7 μm packing diameter). Elution was carried out at 40 °C with
the mobile phases as (A) 10 mM ammonium bicarbonate in water
solution, adjusted to pH 10 with 0.88 ammonia solution, and (B)
acetonitrile using a gradient starting at 3% B, increasing linearly to
95% B over 1.5 min, and remaining at 95% B for 0.4 min. The flow
rate was 1 mL/min. The UV detection was a summed signal from
wavelengths of 210−350 nm. MS analysis was carried out using
alternate-scan positive and negative electrospray. Method B: Kinetex
C8 column (30 mm × 2.1 mm, 1.7 μm packing diameter). Elution
was carried out at rt with the mobile phases as (A) 0.1% formic acid in
water and (B) 0.1% formic acid in acetonitrile using a gradient
starting at 5% B, increasing linearly to 95% B over 2.75 min, and
remaining at 95% B for 0.25 min. The flow rate was 0.65 mL/min.
The UV detection was a summed signal from wavelengths of 210−
400 nm. MS analysis was carried out using alternate-scan positive and
negative electrospray. Method C: Shimadzu 2010 Mass Spectrometer
equipped with an Xtimate C18 column (30 mm × 2.1 mm, 3 μm
packing diameter). Elution was carried out at 50 °C with the mobile
phases as (A) 0.04% trifluoroacetic acid (TFA) in water and (B)
0.02% TFA in acetonitrile using a gradient starting at 0% B, increasing
linearly to 60% B over 0.90 min, and remaining at 60% B for 0.60 min.
The flow rate was 1.2 mL/min. The UV detection was performed at
220 nm. MS analysis was carried out using positive electrospray.
Method D: as method C but using a gradient starting at 10% B,
increasing linearly to 80% B over 0.90 min, and remaining at 80% B
for 0.60 min. Method E: as method C but using a gradient starting at
0% B, increasing linearly to 30% B over 0.90 min, and remaining at
30% B for 0.60 min. Method F: the Waters ZQ Acquity UPLC system
equipped with an CSH C18 column (50 mm × 2.1 mm, 1.7 μm
packing diameter). Elution was carried out at 40 °C with the mobile
phases as (A) 0.1% formic acid in water and (B) 0.1% formic acid in
acetonitrile using a gradient starting at 3% B, increasing linearly to
95% B over 1.5 min, and remaining at 95% B for 0.4 min. The flow
rate was 1 mL/min. The UV detection was a summed signal from
wavelengths of 210 to 350 nm. MS analysis was carried out using
alternate-scan positive and negative electrospray.
profile in dogs, but it exhibited off-target BCP activity and high
clearance in the rat leading to a high predicted human dose. As
part of lead optimization efforts, structural information guided
an exploration at the benzimidazole N1-position and minor
modifications to the 4-methylene THP substituent were
sufficient to abolish BAZ2A activity. In parallel with these
investigations, substituents were included at the 5- and 6-
positions and electron-donating groups were found to increase
activity while also improving metabolic stability. Combination
of the optimized N1-dimethoxypropyl and 5-morpholine
groups resulted in dimethylpyridone benzimidazole 24, a
compound possessing high BRD4 and WB activity, and
favorable physicochemical properties. This 3,5-dimethylpyr-
idone benzimidazole is devoid of BAZ2A activity and possesses
PK properties leading to predicted human doses of <30 mg
and thereby minimizes risk associated with compound-related
attrition. Finally, engagement of the BET mechanism and
immunomodulatory effects were demonstrated in vivo
confirming dimethylpyridone benzimidazole 24 as a candi-
date-quality molecule.
EXPERIMENTAL SECTION
■
Chemistry Methods and Characterization for Compounds
8−26. All commercial chemicals and solvents used were of reagent
grade and used without further purification. Hydrophobic frit
cartridges by ISOLUTE, which contain a frit selectively permeable
to organic solutions, were used for separation of organic phases from
aqueous phases under gravity. ISOLUTE aminopropyl cartridges were
used for scavenging SPE protocols. ISOLUTE C18 cartridges were
used for SPE workup of palladium-catalyzed amination reactions.
Column chromatography was carried out either using manual or
automated flash chromatography systems including a Biotage SP4
using SNAP silica cartridges or a CombiFlash using RediSep silica
cartridges. Preparative high-performance liquid chromatography
(HPLC) and mass-directed autopreparative HPLC (MDAP)
purification were conducted using a Waters ZQ MS using alternate-
scan positive and negative electrospray using either of the following
two LC methods: method A: Either a SunFire C18 column (100 mm
× 19 mm, 5 μm packing diameter) at a 20 mL/min flow rate or a
SunFire C18 column (150 mm × 30 mm, 5 μm packing diameter) at
a 40 mL/min flow rate. Gradient elution was carried out at ambient
temperature, with the mobile phases as (A) water containing 0.1% (v/
v) formic acid and (B) acetonitrile containing 0.1% (v/v) formic acid.
The UV detection was a summed signal from wavelengths of 210 to
350 nm. Method B: either an Xbridge C18 column (100 mm × 19
mm, 5 μm packing diameter) at a 20 mL/min flow rate or an Xbridge
C18 column (150 mm × 30 mm, 5 μm packing diameter) at a 40 mL/
min flow rate. Gradient elution was carried out at ambient
temperature, with the mobile phases as (A) 10 mM ammonium
bicarbonate in water solution, adjusted to pH 10 with 0.88 ammonia
solution, and (B) acetonitrile. The UV detection was a summed signal
from wavelengths of 210 to 350 nm. Microwave irradiation was
carried out using either an Anton Paar Multiwave Pro or a Biotage I60
system. Melting points were measured using a Buchi M-565 automatic
melting point apparatus. IR spectra were obtained on a PerkinElmer
Spectrum 2 spectrometer. 1H NMR spectra were recorded on either a
Bruker AV-400 spectrometer at 400 MHz or a Bruker AV-600
spectrometer at 600 MHz or Varian VNMR400 at 400 MHz. 13C
NMR spectra were recorded on a Bruker AV-600 spectrometer at 150
MHz. NMR spectra were acquired at 293 K, and chemical shifts (δ)
are reported in parts per million (ppm) relative to CHCl3 (δ 7.26),
dimethyl sulfoxide (DMSO; δ 2.50), and MeOH (δ 3.31). Coupling
constants (J) are expressed in hertz (Hz) to the nearest 0.5 Hz.
HRMS were recorded on a Micromass Q-Tof Ultima hybrid
quadrupole time-of-flight mass spectrometer after liquid chromatog-
raphy separation using an Agilent 1100 Liquid Chromatograph
equipped with a Phenomenex Luna C18 reversed phase column (100
2-(4-Hydroxy-3,5-dimethylphenyl)-N-methyl-1-(piperidin-4-yl-
methyl)-1H-benzo[d]imidazole-5-carboxamide Trifluoroacetic Acid
Salt (8). To a solution of 4-fluoro-N-methyl-3-nitrobenzamide (19.8
mg, 0.10 mmol) in EtOH (2 mL) were added tert-butyl 4-
(aminomethyl)piperidine-1-carboxylate (21.4 mg, 0.10 mmol) and
diisopropylethylamine (DIPEA; 52 μL, 0.30 μmol). The reaction
mixture was heated at 80 °C overnight, cooled to rt, and concentrated
to dryness. The resulting crude was dissolved in EtOH (2 mL), and 4-
hydroxy-3,5-dimethylbenzaldehyde (30.0 mg, 0.20 mmol) was added
followed by Na2S2O4 (104 mg, 0.60 mmol) in water (0.5 mL). The
reaction mixture was stirred in a sealed tube at 80 °C for 18 h, cooled
to rt, and concentrated to a small volume. The residue was diluted
with EtOAc (2 mL), and the organic layer was washed with saturated
aqueous NaHCO3 (2 mL). The layers were separated, and the
aqueous layer was extracted with EtOAc (2 × 2 mL). The organics
were combined, dried over Na2SO4, filtered, and concentrated to
dryness to give crude tert-butyl 4-((2-(4-hydroxy-3,5-dimethylphen-
yl)-5-(methylcarbamoyl)-1H-benzo[d]imidazol-1-yl)methyl)-
piperidine-1-carboxylate. This was treated with 50% TFA in
dichloromethane (DCM; 1 mL) for 3 h at rt and then concentrated
to dryness. The crude was purified directly by preparative HPLC
(SunFire C8 OBD Prep Column [30 mm × 50 mm, 5 μm], 40 mL/
min, water/MeCN containing 0.1% TFA, UV detection at 220 nm) to
1
give the title compound (20 mg, 0.05 mmol, 38% yield). H NMR
N
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
Article
(400 MHz, DMSO-d6): δ 9.10 (br s, 1 H), 8.55 (br s, 1 H), 8.19 (s, 1
H), 8.03 (br s, 1 H), 7.89 (m, 2 H), 7.44 (s, 2 H), 4.40 (d, 2 H, J =
7.0 Hz), 3.17−3.10 (m, 2 H), 2.82 (d, 3 H, J = 4.5 Hz), 2.75−2.65
(m, 2 H), 2.27 (s, 6 H), 2.06 (br s, 1 H), 1.54−1.45 (m, 2 H), 1.20−
1.05 (m, 2 H); LC−MS (method B) m/z: 393 [(M + H)+]; Rt: 0.78
min; 95% purity.
tert-Butyl 4-((2-Nitrophenyl)amino)piperidine-1-carboxylate
(11b). A mixture of 1-fluoro-2-nitrobenzene (5 g, 35.4 mmol),
K2CO3 (9.79 g, 70.9 mmol) and tert-butyl 4-aminopiperidine-1-
carboxylate (8.52 g, 42.5 mmol) in MeCN (354 mL) was stirred at
110 °C overnight. To the reaction mixture was added EtOAc and
washed with 10% aqueous citric acid, saturated aqueous NaHCO3,
and brine (×3). The organic was dried with Na2SO4 and concentrated
to give the crude material. The residue was purified by column
chromatography on silica gel eluting with petroleum ether/EtOAc
(10:1 to 3:1) to give the title compound as a yellow solid (11.5 g, 34.2
mmol, 97% yield). LC−MS (method C) m/z: 266 [(M − tBu)+], 344
[(M + Na)+]; Rt: 1.47 min; 96% purity.
tert-Butyl 3-(((2-Nitrophenyl)amino)methyl)azetidine-1-carbox-
ylate (11c). To a solution of 1-fluoro-2-nitrobenzene (5 g, 35.4
mmol) in MeCN (200 mL) were added tert-butyl 3-(aminomethyl)-
azetidine-1-carboxylate (7.92 g, 42.5 mmol) and K2CO3 (9.79 g, 70.9
mmol). The reaction mixture was stirred at 80 °C overnight. The
mixture was filtered, and the filtrate was concentrated in vacuo to give
a residue which was diluted with EtOAc (200 mL). The solution was
washed with brine (3 × 50 mL), dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was purified by flash
column chromatography eluting with 100% EtOAc in petroleum ether
to give the title compound as a bright yellow solid (11.04 g, 34.8
mmol, 98% yield). LC−MS (method D) m/z: 252 [(M − tBu)+], 330
[(M + Na)+]; Rt: 1.10 min; 97% purity.
1-((1-Benzoylpiperidin-4-yl)methyl)-2-(4-hydroxy-3,5-dimethyl-
phenyl)-N-methyl-1H-benzo[d]imidazole-5-carboxamide Trifluoro-
acetic Acid Salt (9). To a solution of benzoic acid (12.2 mg, 0.10
mmol) in dimethylformamide (DMF; 0.5 mL) were added 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluorophosphate (HATU; 38.0 mg, 0.10 mmol) and
DIPEA (52 μL, 0.30 mmol). The reaction mixture was stirred at rt for
5 min and added to a solution of 2-(4-hydroxy-3,5-dimethylphenyl)-
N-methyl-1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazole-5-carboxa-
mide trifluoroacetic acid salt 8 (20 mg, 0.38 mmol) in DMF (0.5 mL).
The reaction mixture was stirred at rt overnight and diluted with
EtOAc (2 mL). The organic layer was washed with saturated aqueous
NaHCO3 (2 mL), and the layers were separated. The aqueous layer
was extracted with EtOAc (2 × 2 mL), and the organics were
combined, dried over Na2SO4, and filtered. The filtrate was
concentrated to dryness, and the crude material was purified by
preparative HPLC (SunFire C8 OBD Prep Column [30 mm × 50
mm, 5 μm], 40 mL/min, water/MeCN containing 0.1% TFA, UV
detection at 220 nm) to give the title compound (13 mg, 0.03 mmol,
1
53% yield). H NMR (400 MHz, DMSO-d6): δ 9.14 (br s, 1 H),
tert-Butyl 3-(((2-Nitrophenyl)amino)methyl)pyrrolidine-1-car-
boxylate (11d). A mixture of 1-fluoro-2-nitrobenzene (3 g, 21.2
mmol), K2CO3 (5.88 g, 42.5 mmol), and tert-butyl 3-(aminomethyl)-
pyrrolidine-1-carboxylate (5.11 g, 25.5 mmol) in MeCN (213 mL)
was stirred at 110 °C overnight. To the reaction mixture was added
EtOAc, and this was washed with 10% aqueous citric acid, saturated
aqueous NaHCO3, and brine (× 3). The organic layer was dried with
Na2SO4 and concentrated to give the crude material. The residue was
purified by column chromatography on silica gel eluting with
petroleum ether/EtOAc (10:1 to 3:1) to give the title compound as
a yellow solid (6.8 g, 21.0 mmol, 99% yield). LC−MS (method D)
m/z: 266 [(M − tBu)+], 344 [(M + Na)+]; Rt: 1.17 min; 99% purity.
tert-Butyl (trans-4-(((2-Nitrophenyl)amino)methyl)cyclohexyl)-
carbamate (11e). To 1-fluoro-2-nitrobenzene (2.6 g, 18.4 mmol)
in MeCN (113 mL) were added tert-butyl (trans-4-(aminomethyl)-
cyclohexyl)carbamate (5.05 g, 22.1 mmol) and K2CO3 (2.55 g, 18.4
mmol). The reaction mixture was stirred at 100 °C. After cooling to
rt, the reaction mixture was filtered on a pad of Celite. The solvent
was washed twice with saturated aqueous NaHCO3 and water and
then dried over Na2SO4 and concentrated in vacuo to give the crude
product as a yellow gum. This was purified by column
chromatography on silica gel eluting with petroleum ether/EtOAc
(5:1 to 4:1) to give the title compound as a yellow gum (5.93 g, 17.0
mmol, 92% yield). LC−MS (method C) m/z: 294 [(M − tBu)+]; Rt:
1.49 min; 100% purity.
tert-Butyl (cis-4-(((2-Nitrophenyl)amino)methyl)cyclohexyl)-
carbamate (11f). A mixture of 1-fluoro-2-nitrobenzene (1.54 g,
10.9 mmol), tert-butyl (cis-4-(aminomethyl)cyclohexyl)carbamate
(2.99 g, 13.1 mmol), and K2CO3 (3.02 g, 21.8 mmol) in MeCN
(72.8 mL) was stirred at 100 °C overnight. The reaction mixture was
filtered and evaporated in vacuo to dryness and purified by column
chromatography on silica gel eluting with petroleum ether/EtOAc
(5:1 to 1:1) to give the title compound as a yellow solid (3.8 g, 10.9
mmol, 100% yield). LC−MS (method D) m/z: 294 [(M − tBu)+]; Rt:
1.22 min; 100% purity.
tert-Butyl 4-(((2-Aminophenyl)amino)methyl)piperidine-1-car-
boxylate (12a). A solution of tert-butyl 4-(((2-nitrophenyl)amino)-
methyl)piperidine-1-carboxylate 11a (50 g, 149 mmol) and Pd/C (1
g, 0.940 mmol) in DMF (300 mL) was stirred at 30 °C overnight
under 1 atm of hydrogen. The reaction mixture was filtered through a
pad of Celite and was washed with EtOAc (3 × 45 mL) and brine.
The combined organics was concentrated to give the title compound
as a dark brown solid (43 g, 140 mmol, 94% yield). LC−MS (method
C) m/z: 206 [(M + H)+]; Rt: 1.13 min; 99% purity.
8.60−8.50 (m, 1 H), 8.16 (s, 1 H), 8.01−7.89 (m, 2 H), 7.46 (s, 2 H),
7.42−7.36 (m, 3 H), 7.31−7.25 (m, 2 H), 4.41 (m, 2 H), 4.37−4.21
(m, 2 H), 3.53−3.32 (m, 2 H), 2.8 (d, 3 H, J = 4.5 Hz), 2.27 (s, 6 H),
2.06−1.93 (m, 1 H), 1.42−1.30 (m, 2 H), 1.11−0.96 (m, 2 H); LC−
MS (method B) m/z: 497 [(M + H)+]; Rt: 1.30 min; 95% purity.
1-((1-Acetylpiperidin-4-yl)methyl)-2-(4-hydroxy-3,5-dimethyl-
phenyl)-N-methyl-1H-benzo[d]imidazole-5-carboxamide Trifluoro-
acetic Acid Salt (10). To a solution of 2-(4-hydroxy-3,5-
dimethylphenyl)-N-methyl-1-(piperidin-4-ylmethyl)-1H-benzo[d]-
imidazole-5-carboxamide trifluoroacetic acid salt 8 (20 mg, 0.38
mmol) in a mixture of tetrahydrofuran (THF; 0.5 mL) and MeCN
(0.5 mL) were added pyridine (40 μL, 0.50 mmol) and acetic
anhydride (28 μL, 0.30 mmol) at 0 °C. The reaction mixture was
stirred at rt overnight and concentrated to dryness. The crude was
partitioned between EtOAc (1 mL) and aqueous NaHCO3 (1 mL),
and the layers were separated. The aqueous layer was extracted with
EtOAc (2 × 1 mL), the organics combined, dried over Na2SO4,
filtered, and concentrated. The material was dissolved in a mixture of
THF (2 mL)/MeOH (0.5 mL) and treated with 2 M LiOH (0.15
mL) for 3 h. The reaction mixture was concentrated to dryness, and
the crude material was purified by preparative HPLC (SunFire C8
OBD Prep Column [30 mm × 50 mm, 5 μm], 40 mL/min, water/
MeCN containing 0.1% TFA, UV detection at 220 nm) to give the
title compound (16 mg, 0.29 mmol, 78% yield). 1H NMR (400 MHz,
DMSO-d6): δ 9.15 (br s, 1 H), 8.61−8.56 (m, 1 H), 8.18 (s, 1 H),
7.97 (d, 1 H, J = 8.0 Hz), 7.93 (d, 1 H, J = 8.0 Hz), 7.47 (s, 2 H),
4.45−4.35 (m, 2 H), 4.22−4.13 (m, 1 H), 3.70−3.60 (m, 1H), 2.82
(d, 3 H, J = 4.5 Hz), 2.39−2.25 (m, 2 H), 2.27 (s, 6 H), 2.00−1.92
(m, 1 H), 1.45−1.35 (m, 1 H), 1.32−1.22 (m, 1 H), 1.10−0.95 (m, 1
H), 0.89−0.75 (m, 1 H); LC−MS (method F) m/z: 435 [(M + H)+];
Rt: 0.52 min; 100% purity.
tert-Butyl 4-(((2-Nitrophenyl)amino)methyl)piperidine-1-carbox-
ylate (11a). A mixture of 1-fluoro-2-nitrobenzene (40 g, 283 mmol),
tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (72.9 g, 340
mmol), and K2CO3 (78 g, 567 mmol) in MeCN (1500 mL) was
stirred at 80 °C overnight. The reaction mixture was washed with 10%
aqueous citric acid, saturated aqueous NaHCO3, and brine (×3). The
organic layer was dried with Na2SO4 and concentrated to give the
crude material. The residue was purified by column chromatography
on silica gel eluting with petroleum ether/EtOAc (10/1 to 3:1, v/v)
to give the title compound as a crude yellow solid (117 g, 337 mmol).
t
LC−MS (method C) m/z: 280 [(M − Bu)+]; Rt: 1.49 min; 97%
purity.
O
DOI: 10.1021/acs.jmedchem.9b01670
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
tert-Butyl 4-((2-Aminophenyl)amino)piperidine-1-carboxylate
(12b). A solution of tert-butyl 4-((2-nitrophenyl)amino)piperidine-
1-carboxylate 11b (11.5 g, 35.8 mmol) and palladium (1.15 g, 1.08
mmol) in DMF (300 mL) was stirred at 30 °C overnight under 1 atm
of hydrogen. The reaction mixture was filtered through a pad of Celite
and was washed with EtOAc (3 × 45 mL) and brine. The combined
organics was concentrated to give the title compound as a dark brown
solid (10.4 g, 33.7 mmol, 94% yield). LC−MS (method C) m/z: 236
give a residue which was purified on silica gel eluting with 1−2%
MeOH in DCM to give the title compound as a yellow solid (5.27 g,
12.3 mmol, 38% yield). LC−MS (method C) m/z: 408 [(M + H)+];
Rt: 1.10 min; 95% purity.
tert-Butyl 3-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)pyrrolidine-1-carboxylate (13d). A solution
of tert-butyl 3-(((2-aminophenyl)amino)methyl)pyrrolidine-1-carbox-
ylate 12d (6.3 g, 21.6 mmol), 4-hydroxy-3,5-dimethylbenzaldehyde
(3.08 g, 20.5 mmol), and Na2S2O5 (5.34 g, 28.1 mmol) in DMF (216
mL) was stirred at 100 °C under nitrogen overnight. The mixture was
added to EtOAc (50 mL), and this was washed with brine (×3). The
organic layer was dried over Na2SO4. The crude was purified by
column chromatography on silica gel eluting with petroleum ether/
EtOAc (20:1 to 3:1) to give the title compound as a pale solid (6.6 g,
15.3 mmol, 71% yield). LC−MS (method C) m/z: 422 [(M + H)+];
Rt: 1.15 min; 97% purity.
t
[(M − Bu)+], 292 [(M + H)+]; Rt: 1.05 min; 94% purity.
tert-Butyl 3-(((2-Aminophenyl)amino)methyl)azetidine-1-car-
boxylate (12c). A solution of tert-butyl 3-(((2-nitrophenyl)amino)-
methyl)azetidine-1-carboxylate 11c (11.04 g, 34.8 mmol) in DMF
(300 mL) was treated with Pd/C (1.0 g, 9.40 mmol). The reaction
mixture was hydrogenated under 30 psi pressure at 30 °C overnight.
The mixture was filtered through a pad of Celite. The solution was
diluted with EtOAc (500 mL), washed with water and brine, dried
over Na2SO4, filtered, and concentrated in vacuo to give the title
compound as a yellow solid (8.89 g, 31.7 mmol, 91% yield). LC−MS
tert-Butyl (trans-4-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-
benzo[d]imidazole-1-yl)methyl)cyclohexyl)carbamate (13e). Step
b: tert-Butyl (trans-4-(((2-nitrophenyl)amino)methyl)cyclohexyl)-
carbamate 11e (5.93 g, 17.0 mmol) was hydrogenated utilizing Pd/
C (0.59 g, 5.54 mmol) under 30 psi pressure in DMF (150 mL) at 30
°C overnight. After cooling to rt, the reaction mixture was filtered and
washed with EtOAc (25 mL). The solution was concentrated in vacuo
to give the crude tert-butyl (trans-4-(((2-aminophenyl)amino)-
methyl)cyclohexyl)carbamate 12e which was used for the next step
directly without purification. Step c: to crude tert-butyl (trans-4-(((2-
aminophenyl)amino)methyl)cyclohexyl)carbamate 12e (5.3 g, 16.6
mmol) in DMF (150 mL) were added 4-hydroxy-3,5-dimethylben-
zaldehyde (2.49 g, 16.6 mmol) and Na2S2O5 (3.78 g, 19.9 mmol).
The reaction mixture was stirred under nitrogen at 100 °C overnight.
Water was added to the reaction mixture, and the aqueous layer was
extracted with DCM (2 × 100 mL). The combined organic layers
were washed with brine and water and then dried over Na2SO4. The
crude product was chromatographed on silica gel eluting with
petroleum ether/EtOAc (3:1, 2:1, 1:1) to give the title compound as a
pale white solid (7 g, 15.4 mmol, 93% overall yield). LC−MS
(method C) m/z: 450 [(M + H)+]; Rt: 1.18 min; 100% purity.
tert-Butyl (cis-4-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo-
[d]imidazol-1-yl)methyl)cyclohexyl)carbamate (13f). A mixture of
tert-butyl (cis-4-(((2-nitrophenyl)amino)methyl)cyclohexyl)-
carbamate 12f (3.48 g, 10.9 mmol), 4-hydroxy-3,5-dimethylbenzalde-
hyde (1.72 g, 11.4 mmol), and Na2S2O5 (2.69 g, 14.1 mmol) in DMF
(150 mL) was stirred under nitrogen at 100 °C overnight. The
reaction mixture was treated with brine, and the organic layer was
washed with brine (×2) and then dried over Na2SO4 and
concentrated in vacuo to give the crude which was purified by
column chromatography on silica gel eluting with petroleum ether/
EtOAc (3:1 to 1:1) to give the title compound as a white solid (4.1 g,
9.1 mmol, 83% yield). LC−MS (method C) m/z: 450 [(M + H)+];
Rt: 1.19 min; 99% purity.
2,6-Dimethyl-4-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazole-
2-yl)phenol Dihydrochloride (14a). A solution of tert-butyl 4-((2-(4-
hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-1-yl)methyl)-
piperidine-1-carboxylate 13a (200 mg, 0.46 mmol) in DCM (2 mL)
was charged with 4 M HCl in MeOH (2 mL, 8.00 mmol). The
reaction was stirred at 50 °C for 1 h. The solvent was removed under
high vacuum to give the title compound as a yellow solid (186 mg,
0.46 mmol, 99% yield). LC−MS (method C) m/z: 336 [(M + H)+];
Rt: 0.82 min; 100% purity.
2,6-Dimethyl-4-(1-(piperidin-4-yl)-1H-benzo[d]imidazole-2-yl)-
phenol Dihydrochloride (14b). A solution of tert-butyl 4-(2-(4-
hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-1-yl)piperidine-
1-carboxylate 13b (3.6 g, 8.54 mmol) and 4 M HCl in MeOH (80
mL, 320 mmol) was stirred at rt for 1 h. The solvent was removed,
and the residue was washed with EtOAc (3 × 5 mL) and evaporated
to give the title compound as a yellow solid (3 g, 7.61 mmol, 89%
yield). 1H NMR (300 MHz, DMSO-d6): δ 9.89−9.71 (m, 1 H), 9.41
(br s, 1 H), 9.03 (d, J = 9.0 Hz, 1 H), 8.63−8.55 (m, 1 H), 7.88−7.79
(m, 1 H), 7.63−7.53 (m, 2 H), 7.40 (s, 2 H), 4.94−4.78 (m, 1 H),
3.12 (q, J = 11.0 Hz, 2 H), 2.93 (q, J = 11.0 Hz, 2 H), 2.30 (s, 6 H),
t
(method C) m/z: 178 [(M − Bu)+]; Rt: 0.83 min; 89% purity.
tert-Butyl 3-(((2-Aminophenyl)amino)methyl)pyrrolidine-1-car-
boxylate (12d). A solution of tert-butyl 3-(((2-nitrophenyl)amino)-
methyl)pyrrolidine-1-carboxylate 11d (6.8 g, 21.2 mmol) and
palladium (0.72 g, 0.68 mmol) in DMF (300 mL) was stirred at 30
°C overnight under 1 atm of hydrogen. The reaction mixture was
filtered through a pad of Celite and was washed with EtOAc (3 × 45
mL) and brine. The combined organic extracts were concentrated to
give the title compound as a dark brown solid (6.3 g, 20.9 mmol, 99%
t
yield). LC−MS (method C) m/z: 192 [(M − Bu)+]; Rt: 1.06 min;
97% purity.
tert-Butyl (cis-4-(((2-Nitrophenyl)amino)methyl)cyclohexyl)-
carbamate (12f). tert-Butyl (cis-4-(((2-nitrophenyl)amino)methyl)-
cyclohexyl)carbamate 11f (3.8 g, 10.9 mmol) was hydrogenated
utilizing Pd/C (0.231 g, 2.18 mmol) under 30 psi pressure in DMF
(150 mL) overnight. The reaction mixture was filtered and evaporated
in vacuo to dryness to give the title compound (3.32 g, 10.2 mmol,
93% yield). LC−MS (method D) m/z: 320 [(M + H)+]; Rt: 0.89 min;
91% purity.
tert-Butyl 4-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)piperidine-1-carboxylate (13a). To a mixture
of tert-butyl 4-(((2-aminophenyl)amino)methyl)piperidine-1-carbox-
ylate 12a (423 mg, 1.385 mmol) and 4-hydroxy-3,5-dimethylbenzal-
dehyde (208 mg, 1.39 mmol) in DMF (3 mL) was added Na2S2O5
(342 mg, 1.80 mmol). The reaction mixture was stirred at 100 °C
overnight. The mixture was cooled and filtered. The mixture was
diluted with EtOAc (30 mL), washed with brine (3 × 10 mL), dried
over Na2SO4, filtered, and concentrated. The residue was purified on
silica gel eluting with 0−3% MeOH in DCM to give the title
compound as a yellow solid (469 mg, 1.07 mmol, 77% yield). LC−
MS (method C) m/z: 436 [(M + H)+]; Rt: 0.97 min; 99% purity.
tert-Butyl 4-(2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)piperidine-1-carboxylate (13b). A solution of tert-
butyl 4-((2-aminophenyl)amino)piperidine-1-carboxylate 12b (10.4
g, 35.7 mmol), 4-hydroxy-3,5-dimethylbenzaldehyde (5.36 g, 35.7
mmol), and Na2S2O5 (8.82 g, 46.4 mmol) in DMF (357 mL) was
stirred at 100 °C under nitrogen overnight. To the mixture was added
EtOAc (50 mL) which was then washed with brine (×3). The organic
layer was dried over Na2SO4. The crude was purified by column
chromatography on silica gel eluting with petroleum ether/EtOAc
(20:1 to 3:1) to give the title compound as a pale solid (10.6 g, 24.8
mmol, 70% yield). LC−MS (method C) m/z: 422 [(M + H)+]; Rt:
1.17 min; 99% purity.
tert-Butyl 3-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)azetidine-1-carboxylate (13c). To a solution
of tert-butyl 3-(((2-aminophenyl)amino)methyl)azetidine-1-carboxy-
late 12c (8.89 g, 32.1 mmol) and 4-hydroxy-3,5-dimethylbenzalde-
hyde (4.81 g, 32.1 mmol) in DMF (80 mL) was added Na2S2O5 (7.92
g, 41.7 mmol). The reaction mixture was stirred at 100 °C overnight.
The mixture was filtered, and the solid was washed with DCM (300
mL). The filtrate was washed with water (3 × 100 mL) and brine (3
× 50 mL), dried over Na2SO4, filtered, and concentrated in vacuo to
P
DOI: 10.1021/acs.jmedchem.9b01670
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2.21 (d, J = 11.0 Hz, 2 H), two piperidine proton signals obscured by
DMSO; LC−MS (method C) m/z: 322 [(M + H)+]; Rt: 0.80 min;
100% purity.
2.42 (m, 1 H), 2.36 (s, 6 H), 2.20−2.14 (m, 1 H), 2.02 (s, 3 H), 1.61
(d, J = 13.5 Hz, 1 H), 1.50 (d, J = 13.0 Hz, 1 H), 1.22−1.11 (m, 1 H),
1.06−0.96 (m, 1 H); LC−MS (method A) m/z: 378 [(M + H)+]; Rt:
0.88 min; 98% purity.
4-(1-(Azetidin-3-ylmethyl)-1H-benzo[d]imidazol-2-yl)-2,6-dime-
thylphenol Dihydrochloride (14c). Batch 1: a solution of tert-butyl 3-
((2-(4-hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-1-yl)-
methyl)azetidine-1-carboxylate 13c (1.0 g, 2.454 mmol) in 4 M HCl
in MeOH (20 mL, 80 mmol) was stirred at 45 °C for 1 h. The solvent
was removed in vacuo to give the crude product. Batch 2: a solution
of tert-butyl 3-((2-(4-hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)azetidine-1-carboxylate 13c (5.27 g, 12.93
mmol) in 4 M HCl in MeOH (100 mL, 400 mmol) was stirred at
45 °C for 1 h. The solvent was removed in vacuo to give the crude
product. Both batches were combined and washed with 5% MeOH in
DCM to afford the title compound as a pink solid (5.1 g, 12.2 mmol,
79% yield). LC−MS (method E) m/z: 308 [(M + H)+]; Rt: 0.81 min;
91% purity.
1-(4-(2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-1-
yl)piperidin-1-yl)ethanone (15b). A mixture of 2,6-dimethyl-4-(1-
(piperidin-4-yl)-1H-benzo[d]imidazol-2-yl)phenol dihydrochloride
14b (79 mg, 200 μmol), NEt3 (24.29 mg, 240 μmol), BOP (133
mg, 300 μmol), and AcOH (48.0 mg, 800 μmol) in DCM (2 mL) was
stirred at rt overnight. The reaction mixture was purified by
preparative HPLC to give the title compound as a pale white gum
1
(14 mg, 0.37 mmol, 19% yield). H NMR (400 MHz, MeOH-d4): δ
7.74−7.66 (m, 2 H), 7.32−7.30 (m, 2 H), 7.25 (s, 2 H), 4.12 (d, J =
14.0 Hz, 1 H), 3.24−3.15 (m, 2 H), 2.70−2.42 (m, 3 H), 2.32 (s, 6
H), 2.98 (s, 3 H), 2.01 (br s, 2 H), 1.33−1.30 (m, 1 H); LC−MS
(method A) m/z: 364 [(M + H)+]; Rt: 0.83 min; 100% purity.
1-(3-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-
1-yl)methyl)azetidin-1-yl)ethanone (15c). A mixture of 4-(1-
(azetidin-3-ylmethyl)-1H-benzo[d]imidazol-2-yl)-2,6-dimethylphenol
14c (61.5 mg, 0.20 mmol), AcOH (14.4 mg, 0.24 mmol), BOP (133
mg, 0.30 mmol), and NEt3 (81 mg, 0.80 mmol) in DCM (2 mL) was
stirred at 25 °C for 20 h. The reaction mixture was filtered and
evaporated in vacuo to dryness and purified by preparative HPLC to
give the title compound as a yellow gum (30 mg, 0.09 mmol, 43%
yield). 1H NMR (400 MHz, MeOH-d4): δ 7.69−7.66 (m, 2 H),
7.40−7.32 (m, 4 H), 4.70 (d, J = 7.5 Hz, 2 H), 4.11 (t, J = 8.5 Hz, 1
H), 3.88 (t, J = 9.0 Hz, 1 H), 3.73 (dd, J = 9.0, 5.5 Hz, 1 H), 3.54 (dd,
J = 10.0, 5.5 Hz, 1 H), 3.08−3.01 (m, 1 H), 2.34 (s, 6 H), 1.73 (s, 3
H); LC−MS (method A) m/z: 350 [(M + H)+]; Rt: 0.78 min; 98%
purity.
2,6-Dimethyl-4-(1-(pyrrolidin-3-ylmethyl)-1H-benzo[d]-
imidazole-2-yl)phenol Dihydrochloride (14d). A solution of tert-
butyl 3-((2-(4-hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-1-
yl)methyl)pyrrolidine-1-carboxylate 13d (1 g, 2.37 mmol) and 4 M
HCl in MeOH (20 mL, 80 mmol) was stirred at rt for 1 h. The
solvent was removed, and the residue was washed with EtOAc (3 × 5
mL) and evaporated to give the title compound as a brown solid (0.87
1
g, 2.21 mmol, 93% yield). H NMR (300 MHz, MeOH-d4): δ 8.16−
8.09 (m, 1 H), 7.88−7.81 (m, 1 H), 7.75−7.64 (m, 2 H), 7.57 (s, 2
H), 4.85 (d, J = 6.5 Hz, 2 H), 3.43−3.34 (m, 1 H), 3.28−3.20 (m, 1
H), 3.20−3.08 (m, 1 H), 3.00−2.81 (m, 2 H), 2.37 (s, 6 H), 2.00 (d, J
= 4.5 Hz, 1 H), 1.68−1.51 (m, 1 H); LC−MS (method C) m/z: 322
[(M + H)+]; Rt: 0.79 min; 100% purity.
4-(1-((trans-4-Aminocyclohexyl)methyl)-1H-benzo[d]imidazol-2-
yl)-2,6-dimethylphenol Dihydrochloride (14e). A suspension of tert-
butyl (trans-4-((2-(4-hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)cyclohexyl)carbamate 13e (1 g, 2.22 mmol) in
4 M HCl in MeOH (20 mL, 80 mmol) and MeOH (20 mL) was
stirred at rt for 3 h. The reaction mixture was evaporated in vacuo to
give the title compound as a yellow gum (0.7 g, 1.66 mmol, 75%
yield). 1H NMR (400 MHz, MeOH-d4): δ 8.05−8.00 (m, 1 H),
7.85−7.80 (m, 1 H), 7.71−7.66 (m, 2 H), 7.52 (s, 2 H), 4.54 (d, J =
7.5 Hz, 2 H), 3.04−2.94 (m, 1 H), 2.36 (s, 6 H), 1.96 (d, J = 13.0 Hz,
2 H), 1.93−1.86 (m, 1 H), 1.65 (d, J = 13.0 Hz, 2 H), 1.26 (q, J =
13.0 Hz, 2 H), 1.06 (q, J = 13.0 Hz, 2 H); LC−MS (method C) m/z:
350 [(M + H)+]; Rt: 0.84 min; 99% purity.
1-(3-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-
1-yl)methyl)pyrrolidin-1-yl)ethanone (15d). A mixture of 2,6-
dimethyl-4-(1-(pyrrolidin-3-ylmethyl)-1H-benzo[d]imidazol-2-yl)-
phenol dihydrochloride 14d (80 mg, 0.20 mmol), AcOH (14.6 mg,
0.24 mmol), BOP (135 mg, 0.30 mmol), and NEt3 (82 mg, 0.81
mmol) in DCM (2 mL) was stirred at rt under nitrogen overnight.
The reaction mixture was evaporated in vacuo, and the crude was
purified by preparative HPLC to give the title compound as a pale
1
white gum (46 mg, 0.12 mmol, 61% yield). H NMR (400 MHz,
MeOH-d4): δ 7.70−7.61 (m, 2 H), 7.38−7.29 (m, 4 H), 4.47 (dd, J =
12.0, 8.0 Hz, 2 H), 3.30−3.24 (m, 3 H), 3.02−2.98 (m, 1 H), 2.78−
2.67 (m, 1 H), 2.33 (s, 6 H), 1.91−1.83 (m, 4 H), 1.63−1.61 (m, 1
H); LC−MS (method A) m/z: 364 [(M + H)+]; Rt: 0.81 min; 100%
purity.
4-(1-((cis-4-Aminocyclohexyl)methyl)-1H-benzo[d]imidazol-2-
yl)-2,6-dimethylphenol Dihydrochloride (14f). A mixture of tert-
butyl (cis-4-((2-(4-hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)cyclohexyl)carbamate 13f (1 g, 2.22 mmol)
and 4 M HCl in MeOH (20 mL, 80 mmol) in MeOH (10 mL) was
stirred at rt overnight. The reaction mixture was evaporated in vacuo
to give the title compound as a pale green solid (0.73 g, 1.71 mmol,
77% yield). 1H NMR (400 MHz, MeOH-d4): δ 8.09−8.00 (m, 1 H),
7.86−7.80 (m, 1 H), 7.72−7.63 (m, 2 H), 7.55 (s, 2 H), 4.64 (d, J =
7.5 Hz, 2 H), 3.29−3.26 (m, 1 H), 2.37 (s, 6 H), 2.15−2.04 (m, 1 H),
1.74−1.58 (m, 4 H), 1.56−1.45 (m, 2 H), 1.43−1.30 (m, 2 H); LC−
MS (method C) m/z: 350 [(M + H)+]; Rt: 0.83 min; 99% purity.
1-(4-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]imidazol-
1-yl)methyl)piperidin-1-yl)ethanone Hydrochloride (15a). To a
suspension of 2,6-dimethyl-4-(1-(piperidin-4-ylmethyl)-1H-benzo[d]-
imidazol-2-yl)phenol dihydrochloride 14a (30 mg, 0.073 mmol) in
DCM (0.5 mL) was added NEt3 (0.041 mL, 0.29 mmol), and then,
the mixture was stirred at rt for 5 min. To the solution were added
AcOH (5.3 mg, 0.09 mmol) and (benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate (BOP, 48.7 mg,
0.11 mmol), and then, the mixture was stirred at 30 °C for 3 h. The
mixture was concentrated in vacuo to give a residue which was
purified by preparative HPLC to give the title compound as a
N-(trans-4-((2-(4-hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)cyclohexyl)acetamide (15e). A mixture of 4-
(1-((trans-4-aminocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)-
2,6-dimethylphenol dihydrochloride 14e (80 mg, 0.19 mmol), AcOH
(13.7 mg, 0.23 mmol), BOP (126 mg, 0.28 mmol), and NEt3 (77 mg,
0.76 mmol) in DCM (2 mL) was stirred at rt under nitrogen
overnight. The reaction mixture was evaporated in vacuo to give the
crude, which was purified by preparative HPLC to give the title
1
compound as a pale white gum (28 mg, 0.070 mmol, 37% yield). H
NMR (400 MHz, MeOH-d4): δ 7.67−7.65 (m, 1 H), 7.60−7.58 (m,
1 H), 7.36−7.28 (m, 4 H), 4.28 (d, J = 7.5 Hz, 2 H), 3.51−3.43 (m, 1
H), 2.89 (d, J = 2.0 Hz, 1 H), 2.33 (s, 6 H), 1.86 (s, 3 H), 1.81−1.73
(m, 3 H), 1.47−1.43 (m, 2 H), 1.08−0.90 (m, 4 H); LC−MS
(method F) m/z: 392 [(M + H)+]; Rt: 0.55 min; 100% purity.
N-(cis-4-((2-(4-Hydroxy-3,5-dimethylphenyl)-1H-benzo[d]-
imidazol-1-yl)methyl)cyclohexyl)acetamide (15f). A mixture of 4-
(1-((cis-4-aminocyclohexyl)methyl)-1H-benzo[d]imidazol-2-yl)-2,6-
dimethylphenol dihydrochloride 14f (69.9 mg, 0.20 mmol), AcOH
(14.4 mg, 0.24 mmol), BOP (133 mg, 0.30 mmol), and NEt3 (81 mg,
0.80 mmol) in DCM (1 mL) was stirred at rt over the weekend. The
reaction mixture was evaporated in vacuo to give the crude product
which was purified by preparative HPLC to give the title compound
1
colorless gum (26 mg, 0.06 mmol, 43% yield). H NMR (400 MHz,
1
(19 mg, 0.47 mmol, 24% yield). H NMR (400 MHz, MeOH-d4): δ
MeOH-d4): δ 8.03−8.00 (m, 1 H), 7.83−7.80 (m, 1 H), 7.71−7.65
(m, 2 H), 7.52 (s, 2 H), 4.57 (dd, J = 7.0, 3.0 Hz, 2 H), 4.41 (d, J =
13.5 Hz, 1 H), 3.84 (d, J = 14.0 Hz, 1 H), 3.00−2.93 (m, 1 H), 2.48−
7.68−7.65 (m, 1 H), 7.60 (d, J = 7.0 Hz, 1 H), 7.36−7.29 (m, 4 H),
4.34 (d, J = 7.5 Hz, 2 H), 3.80 (br s, 1 H), 2.33 (s, 6 H), 1.97−1.91
Q
DOI: 10.1021/acs.jmedchem.9b01670
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(m, 4 H), 1.50−1.30 (m, 6 H), 1.22−1.18 (m, 2 H); LCMS (method
F) m/z: 392 [(M + H)+]; Rt: 0.55 min; 100% purity.
(500 mg, 4.06 mmol), and Na2S2O5 (1.16 g, 6.1 mmol) in DMF
(20 mL) was stirred at 100 °C overnight. The reaction mixture was
treated with water and extracted with DCM. The organic layer was
washed with saturated aqueous NaHCO3 solution (×2) and water
and then dried over Na2SO4 and concentrated in vacuo to give tert-
butyl 4-((2-(6-oxo-1,6-dihydropyridin-3-yl)-1H-benzo[d]imidazol-1-
yl)methyl)piperidine-1-carboxylate as a pale yellow solid (1.44 g,
3.5 mmol, 86% yield). LC−MS (method C) m/z: 409 [(M + H)+];
Rt: 1.06 min; 99% purity. Step a (ii): a mixture of tert-butyl 4-((2-(6-
oxo-1,6-dihydropyridin-3-yl)-1H-benzo[d]imidazol-1-yl)methyl)-
piperidine-1-carboxylate (1.44 g, 3.53 mmol) and 6 M HCl in MeOH
(30 mL) was stirred at rt overnight. The reaction mixture was
evaporated in vacuo to give the title compound as a white solid (1.3 g,
3.4 mmol, 96% yield). LC−MS (method C) m/z: 309 [(M + H)+];
Rt: 0.28 min; 99% purity.
2-Hydroxy-1-(4-((2-(4-hydroxy-3,5-dimethylphenyl)-1H-benzo-
[d]imidazol-1-yl)methyl)piperidin-1-yl)ethanone (15g). A mixture
of 2,6-dimethyl-4-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazol-2-
yl)phenol hydrochloride 14a (60 mg, 0.16 mmol), 2-hydroxyacetic
acid (18.4 mg, 0.24 mmol), BOP (107 mg, 0.24 mmol), and NEt3
(0.09 mL, 0.65 mmol) in DCM (1 mL) under nitrogen was stirred at
rt overnight. The crude was purified by preparative thin-layer
chromatography (TLC) (DCM/MeOH 20:1) to give the title
1
compound as a pale white gum (24 mg, 0.06 mmol, 36% yield). H
NMR (400 MHz, MeOH-d4): δ 7.68−7.60 (m, 2 H), 7.36−7.27 (m,
4 H), 4.37−4.31 (m, 3 H), 4.13 (d, J = 3.5 Hz, 2 H), 3.63−3.57 (m, 1
H), 2.88−2.78 (m, 1 H), 2.55−2.46 (m, 1 H), 2.32 (s, 6 H), 2.13−
2.01 (m, 1H), 1.45−1.39 (m, 2 H), 1.13−0.92 (m, 2 H); LC−MS
(method C) m/z: 394 [(M + H)+]; Rt: 0.94 min; 97% purity.
1,3-Dimethyl-5-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazol-
2-yl)pyridin-2(1H)-one Dihydrochloride (18a). Step a (i): a solution
of tert-butyl 4-(((2-aminophenyl)amino)methyl)piperidine-1-carbox-
ylate 12a (2.1 g, 6.9 mmol), 1,5-dimethyl-6-oxo-1,6-dihydropyridine-
3-carbaldehyde (1.14 g, 7.6 mmol), and Na2S2O5 (1.70 g, 8.9 mmol)
in DMF (50 mL) was stirred at 100 °C under nitrogen for 6 h. EtOAc
(30 mL) was added to the mixture which was washed with brine
(×3). The organic layer was dried over Na2SO4 and concentrated in
vacuo to give tert-butyl 4-((2-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-
3-yl)-1H-benzo[d]imidazol-1-yl)methyl)piperidine-1-carboxylate as a
brown oil (2.86 g, 6.4 mmol, 92% yield). LC−MS (method C) m/z:
437 [(M + H)+]; Rt: 1.15 min; 97% purity. Step a (ii): a mixture of
tert-butyl 4-((2-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1H-
benzo[d]imidazol-1-yl)methyl)piperidine-1-carboxylate (2.8 g, 6.4
mmol) and 4 M HCl in MeOH (15 mL, 60.0 mmol) in MeOH
(15 mL) was stirred at 25 °C for 2 h. The solvent was removed in
vacuo to give the title compound as a pale solid (2.44 g, 5.9 mmol,
93% yield). LC−MS (method E) m/z: 337 [(M + H)+]; Rt: 0.84 min;
100% purity.
3-Methyl-5-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazol-2-yl)-
pyridin-2(1H)-one Dihydrochloride (18b). Step a (i): to a solution of
tert-butyl 4-(((2-aminophenyl)amino)methyl)piperidine-1-carboxy-
late 12a (1.03 g, 3.4 mmol) and 5-methyl-6-oxo-1,6-dihydropyr-
idine-3-carbaldehyde (640 mg, 3.36 mmol) in DMF (10 mL) was
added Na2S2O5 (830 mg, 4.37 mmol). The reaction mixture was
stirred at 100 °C overnight. The mixture was filtered, and the solid
was washed with EtOAc (30 mL). The filtrate was washed with water
(3 × 10 mL) and brine (3 × 10 mL), dried over Na2SO4, filtered, and
concentrated in vacuo to give the residue which was purified on silica
gel eluting with 1−4% MeOH in DCM to give tert-butyl 4-((2-(5-
methyl-6-oxo-1,6-dihydropyridin-3-yl)-1H-benzo[d]imidazol-1-yl)-
methyl)piperidine-1-carboxylate as a brown solid (1.17 g, 2.1 mmol,
63% yield). LC−MS (method C) m/z: 423 [(M + H)+]; Rt: 1.13 min;
76% purity. Step a (ii): a solution of tert-butyl 4-((2-(5-methyl-6-oxo-
1,6-dihydropyridin-3-yl)-1H-benzo[d]imidazol-1-yl)methyl)-
piperidine-1-carboxylate (1.17 g, 2.1 mmol) in 4 M HCl in MeOH
(30 mL, 120 mmol) was stirred at rt for 2 h. The solvent was removed
in vacuo to give the title compound as a gray solid (1.04 g, 2.1 mmol,
100% yield). LC−MS (method C) m/z: 323 [(M + H)+]; Rt: 0.52
min; 80% purity.
5-(1-((1-(2-Hydroxyacetyl)piperidin-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (19a). A mixture of
1,3-dimethyl-5-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazol-2-yl)-
pyridin-2(1H)-one dihydrochloride 18a (2.44 g, 6.0 mmol), 2-
hydroxyacetic acid (0.97 g, 12.8 mmol), DIPEA (3.31 g, 25.6 mmol),
1H-benzo[d][1,2,3]triazol-1-ol (HOBt, 1.08 g, 7.0 mmol), and N1-
((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine
(EDC)·HCl (1.35 g, 7.0 mmol) in DCM (60 mL) was stirred at 25
°C overnight. The reaction mixture was purified by column
chromatography on silica gel eluting with DCM/MeOH (200:1 to
50:1). This was further purified by preparative HPLC to give the title
compound as a pale solid (240 mg, 0.58 mmol, 10% yield). 1H NMR
(300 MHz, MeOH-d4): δ 8.03 (d, J = 2.0 Hz, 1 H), 7.76−7.71 (m, 1
H), 7.67 (d, J = 7.5 Hz, 1 H), 7.63 (d, J = 7.5 Hz, 1 H), 7.39−7.28
(m, 2 H), 4.40 (d, J = 12.5 Hz, 1 H), 4.30 (d, J = 7.5 Hz, 2 H), 4.23−
4.07 (m, 2 H), 3.68 (s, 3 H), 3.63 (d, J = 12.5 Hz, 1 H), 2.85 (t, J =
12.5 Hz, 1 H), 2.51 (t, J = 12.5 Hz, 1 H), 2.22 (s, 3 H), 2.18−2.04
(m, 1 H), 1.45 (d, J = 12.5 Hz, 2 H), 1.25−0.99 (m, 2 H); LC−MS
(method C) m/z: 395 [(M + H)+]; Rt: 0.87 min; 95% purity.
5-(1-((1-(2-Hydroxyacetyl)piperidin-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)-3-methylpyridin-2(1H)-one (19b). A mixture of 3-
methyl-5-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazol-2-yl)-
pyridin-2(1H)-one 18b (600 mg, 1.49 mmol) in DCM (10 mL) was
treated with DIPEA (962 mg, 7.44 mmol), 2-hydroxyacetic acid (226
mg, 2.98 mmol), HOBt (274 mg, 1.79 mmol), and EDC·HCl (571
mg, 2.98 mmol) and then stirred at rt under a nitrogen atmosphere
overnight. The mixture was concentrated in vacuo to give the residue
which was purified by preparative TLC (MeOH/DCM = 1:15) to
give the impure material. This was repurified by preparative HPLC
and dried under high vacuum to give the title compound as a white
solid (126 mg, 0.31 mmol, 21% yield). 1H NMR (400 MHz, MeOH-
d4): δ 8.14 (d, J = 2.0 Hz, 1 H), 8.09−8.02 (m, 1 H), 7.88−7.81 (m, 2
H), 7.74−7.66 (m, 2 H), 4.52 (d, J = 5.5 Hz, 2 H), 4.45 (d, J = 12.5
Hz, 1 H), 4.26−4.13 (m, 2 H), 3.69 (d, J = 12.5 Hz, 1 H), 2.93 (t, J =
12.5 Hz, 1 H), 2.57 (t, J = 12.5 Hz, 1 H), 2.28−2.23 (m, 1 H), 2.22
(s, 3 H), 1.61 (t, J = 15.0 Hz, 2 H), 1.34−1.10 (m, 2 H); LC−MS
(method C) m/z: 381 [(M + H)+]; Rt: 0.84 min; 93% purity.
5-(1-((1-(2-Hydroxyacetyl)piperidin-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)-1-methylpyridin-2(1H)-one (19c). A mixture of 1-
methyl-5-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazol-2-yl)-
pyridin-2(1H)-one 18c (500 mg, 1.55 mmol), 2-hydroxyacetic acid
(236 mg, 3.10 mmol), DIPEA (10.5 mL, 7.8 mmol), HOBt (285 mg,
1.86 mmol), and EDC·HCl (595 mg, 3.10 mmol) in DCM (10 mL)
was stirred at rt overnight. The reaction mixture was filtered,
evaporated in vacuo to dryness, and purified by preparative TLC
(DCM/MeOH = 20:1) to give the title compound (62 mg, 0.16
mmol, 10% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.27 (d, J = 2.5
Hz, 1 H), 7.84 (dd, J = 9.5, 2.5 Hz, 1 H), 7.68 (d, J = 7.5 Hz, 1 H),
7.64 (d, J = 7.5 Hz, 1 H), 7.30−7.20 (m, 2 H), 6.54 (d, J = 9.5 Hz, 1
H), 4.38 (t, J = 5.0 Hz, 1 H), 4.32−4.18 (m, 3 H), 4.07−3.93 (m, 2
H), 3.63−3.51 (m, 4 H), 2.78 (t, J = 12.5 Hz, 1 H), 2.44 (t, J = 12.5
Hz, 1 H), 2.06−1.93 (m, 1 H), 1.34 (t, J = 12.0 Hz, 2 H), 1.11 (q, J =
12.0 Hz, 1 H), 0.97 (q, J = 12.0 Hz, 1 H); LC−MS (method F) m/z:
381 [(M + H)+]; Rt: 0.42 min; 100% purity.
1-Methyl-5-(1-(piperidin-4-ylmethyl)-1H-benzo[d]imidazol-2-yl)-
pyridin-2(1H)-one (18c). Step b: a solution of tert-butyl 4-(((2-
aminophenyl)amino)methyl)piperidine-1-carboxylate 12a (1.8 g, 5.9
mmol) and 1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid
(0.99 g, 6.48 mmol) in polyphosphoric acid (PPA) (15 g) was
stirred at 180 °C under a nitrogen atmosphere for 2 h. The reaction
was quenched by saturated aqueous NaHCO3 and purified by
preparative HPLC to give the title compound as a pale solid (1.2 g,
1.9 mmol). LC−MS (method E) m/z: 323 [(M + H)+]; Rt: 0.74 min;
59% purity.
5-(1-(Piperidin-4-ylmethyl)-1H-benzo[d]imidazol-2-yl)pyridin-
2(1H)-one Dihydrochloride (18d). Step a (i): a mixture of tert-butyl
4-(((2-aminophenyl)amino)methyl)piperidine-1-carboxylate 12a
(1.24 g, 4.06 mmol), 6-oxo-1,6-dihydropyridine-3-carbaldehyde
R
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
Article
5-(1-((1-(2-Hydroxyacetyl)piperidin-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)pyridin-2(1H)-one (19d). A mixture of 5-(1-(piperidin-
4-ylmethyl)-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one dihydro-
chloride 18d (500 mg, 1.31 mmol), DIPEA (847 mg, 6.56 mmol),
HOBt (241 mg, 1.57 mmol), EDC·HCl (503 mg, 2.62 mmol), and 2-
hydroxyacetic acid (199 mg, 2.62 mmol) in DCM (10 mL) was
stirred at rt overnight. The reaction mixture was purified by
preparative HPLC and then purified twice further by preparative
TLC (DCM/MeOH = 20:1) to give the title compound as a yellow
using silica gel 100−200 mesh, eluting with EtOAc and then 5%
MeOH in DCM. The collected fractions were concentrated under
reduced pressure. The material was purified by column chromatog-
raphy using silica gel 230−400 mesh, eluting with 2% MeOH in
DCM. The collected fractions were concentrated under reduced
pressure. The material was washed with Et2O (2 × 10 mL), decanted,
and dried under reduced pressure to afford batch 2 (900 mg). Both
batches were combined and purified by flash chromatography
(column: Grace; silica 40 mm; 24 g cartridge). Pure fractions were
collected and concentrated under reduced pressure to afford the title
compound as a white solid (1.2 g, 3.6 mmol, 28% combined yield).
mp 158 °C; IR (solid) ν (cm−1): 2918, 2840, 1654, 1619, 1459, 1091;
1H NMR (600 MHz, DMSO-d6): δ 8.13 (s, 1 H), 7.74 (s, 1 H), 7.68
(d, J = 7.5 Hz, 1 H), 7.62 (d, J = 7.5 Hz, 1 H), 7.26 (t, J = 7.5 Hz, 1
H), 7.21 (t, J = 7.5 Hz, 1 H), 4.28 (d, J = 7.0 Hz, 2 H), 3.72 (dd, J =
11.5, 3.5 Hz, 2 H), 3.56 (s, 3 H), 3.11 (td, J = 11.5, 2.0 Hz, 2 H), 2.11
(s, 3 H), 1.96 (ddd, J = 11.0, 7.0, 4.0 Hz, 1 H), 1.24−1.12 (m, 4 H);
13C NMR (150 MHz, DMSO-d6): δ 162.3, 150.9, 142.8, 138.8, 137.2,
1
solid (56 mg, 0.15 mmol, 12% yield). H NMR (400 MHz, DMSO-
d6): δ 7.90−7.82 (m, 2 H), 7.66 (d, J = 7.5 Hz, 1 H), 7.63 (d, J = 7.5
Hz, 1 H), 7.29−7.19 (m, 2 H), 6.49 (d, J = 9.5 Hz, 1 H), 4.39 (t, J =
5.0 Hz, 1 H), 4.31−4.14 (m, 3 H), 4.08−3.92 (m, 2 H), 3.58 (d, J =
12.5 Hz, 1 H), 2.79 (t, J = 12.5 Hz, 1 H), 2.44 (t, J = 12.5 Hz, 1 H),
2.06−1.92 (m, 1 H), 1.36 (t, J = 12.5 Hz, 2 H), 1.11 (q, J = 12.0 Hz, 1
H), 0.97 (q, J = 12.0 Hz, 1 H), NH not resolved; LC−MS (method
F) m/z: 367 [(M + H)+]; Rt: 0.40 min; 97% purity.
1,3-Dimethyl-5-(1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)pyridin-2(1H)-one (20a). Step a: batch 1:
(tetrahydro-2H-pyran-4-yl)methanamine (0.26 mL, 2.13 mmol) was
added to a solution of 1-fluoro-2-nitrobenzene (0.11 mL, 1.06 mmol)
and DIPEA (0.56 mL, 3.19 mmol) in NMP (1 mL) at rt in a
microwave vial. The vial was sealed and heated in a microwave at 120
°C for 30 min. Once cooled, the reaction mixture was diluted with
EtOAc (5 mL) and the organic layer was extracted with water (4 × 5
mL). The aqueous phase was then back extracted with EtOAc (4 × 5
mL). The combined organic layers were dried over a hydrophobic frit
before being concentrated in vacuo to give an orange oil (∼70 mg).
Batch 2: (tetrahydro-2H-pyran-4-yl)methanamine (3.5 mL, 28.3
mmol) was added to a solution of 1-fluoro-2-nitrobenzene (1.5 mL,
14.1 mmol) and DIPEA (5.0 mL, 28.3 mmol) in NMP (10 mL) at rt
in a microwave vial. The vial was sealed and heated in a microwave at
120 °C for 30 min. Once cooled the reaction mixture was diluted with
EtOAc (50 mL) and the organic layer extracted with water (4 × 50
mL). The aqueous phase was then back extracted with EtOAc (4 × 50
mL). The combined organic layers were dried over a hydrophobic frit
before being concentrated in vacuo to give an orange oil. Both batches
were combined, loaded in DCM, and purified with a CombiFlash RF+
40 g silica cartridge using a gradient of 0−15% EtOAc in cyclohexane
over 10 column volumes and then held at 15% for further 10 column
volumes. The relevant fractions were combined and concentrated in
vacuo to give 2-nitro-N-((tetrahydro-2H-pyran-4-yl)methyl)aniline as
136.5, 128.3, 122.7, 122.3, 119.3, 111.6, 108.8, 66.8 (2C), 49.8, 37.9
(2C), 35.6, 30.4, 17.4; LC−MS (method F) m/z: 338 [(M + H)+];
Rt: 0.52 min; 100% purity. HRMS: [(M + H)+] calcd for
C20H24N3O2, 338.1869; found, 338.1868.
1,3-Dimethyl-5-(1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-
benzo[d]imidazole-2-yl)pyridin-2(1H)-one (20b and 20b′). Step a:
1-fluoro-2-nitrobenzene (0.11 mL, 1.04 mmol), 1-(tetrahydro-2H-
pyran-4-yl)ethanamine hydrochloride (250 mg, 1.51 mmol), and
DIPEA (0.8 mL, 4.58 mmol) were suspended in DMSO (1 mL)
within a sealed vessel and heated in a microwave for 20 min at 120
°C. The reaction mixture was allowed to cool to rt. The reaction
vessel was sealed and heated within a microwave for a further 20 min
at 120 °C. The reaction mixture was allowed to cool to rt. The solvent
was removed under a stream of nitrogen, and the residue was taken up
into EtOAc (10 mL), diluted with water (10 mL), and extracted with
EtOAc (3 × 10 mL). The combined organic extracts were dried over
a hydrophobic frit, and the filtrate was purified by normal phase
column chromatography on an 80 g silica column and eluted with 0−
70% cyclohexane in EtOAc over 10 column volumes. The desired
fractions were combined, and the solvent was removed in vacuo to
give a yellow oil which was dried in a vacuum oven overnight to yield
crude racemic 2-nitro-N-(1-(tetrahydro-2H-pyran-4-yl)ethyl)aniline
(378 mg, 1.51 mmol). 1H NMR (400 MHz, CDCl3): δ 8.18 (dd, J =
8.5, 1.5 Hz, 1 H), 8.12 (d, J = 8.0 Hz, 1 H), 7.42 (ddd, J = 8.5, 7.0, 1.5
Hz, 1 H), 6.86 (d, J = 8.5 Hz, 1 H), 6.62 (ddd, J = 8.5, 7.0, 1.5 Hz, 1
H), 4.07−4.00 (m, 2 H), 3.63−3.52 (m, 1 H), 3.44−3.35 (m, 2 H),
1.83−1.73 (m, 2 H), 1.70−1.63 (m, 1 H), 1.55−1.39 (m, 2 H), 1.27
(d, J = 6.5 Hz, 3 H); LC−MS (method A) m/z: 251 [(M + H)+]; Rt:
1.17 min; 100% purity. Step c (i): racemic 2-nitro-N-(1-(tetrahydro-
2H-pyran-4-yl)ethyl)aniline (142 mg, 0.567 mmol) was dissolved in
EtOH (10 mL) and added to 5% Pd/C (15 mg, 0.007 mmol). The
reaction mixture was stirred at rt under an atmosphere of hydrogen
for 6 h. The reaction mixture was filtered over Celite and washed with
EtOH (30 mL). The solvent was removed in vacuo to give racemic
N1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)benzene-1,2-diamine as a pur-
1
an orange oil (2.3 g, 9.6 mmol, 63% yield). H NMR (400 MHz,
CDCl3): δ 8.18 (dd, J = 8.5, 1.5 Hz, 1 H), 8.21−8.12 (m, 1 H), 7.45
(ddd, J = 8.5, 7.0, 1.5 Hz, 1 H), 6.85 (dd, J = 8.5, 1.5 Hz, 1 H), 6.66
(ddd, J = 8.5, 7.0, 1.5 Hz, 1 H), 4.03 (dd, J = 11.5, 4.0 Hz, 2 H), 3.43
(ddd, J = 12.0, 11.5, 2.0 Hz, 2 H), 3.22 (dd, J = 6.5, 5.5 Hz, 2 H),
2.02−1.90 (m, 1 H), 1.76 (ddd, J = 12.0, 4.0, 2.0 Hz, 2 H), 1.49−1.38
(m, 2 H); LC−MS (method A) m/z: 237 [(M + H)+]; Rt: 1.07 min;
96% purity. Step b (i): batch 1: to a mixture of 2-nitro-N-
((tetrahydro-2H-pyran-4-yl)methyl)aniline (1.5 g, 6.4 mmol), 1,5-
dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde (0.96 g, 6.35
mmol) and Na2S2O4 (2.0 g, 11.4 mmol) in EtOH (2 mL) and
water (1 mL) were added in a sealed tube and stirred at 100 °C for 16
h. The reaction mixture was diluted with water (100 mL) and
extracted with EtOAc (3 × 100 mL). The organic layer was washed
with brine (250 mL), separated, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The crude material was purified
by column chromatography using silica gel 100−200 mesh, eluting
with EtOAc and then 2% MeOH in DCM. The collected fractions
were concentrated under reduced pressure to afford batch 1 (560
mg). Batch 2: to a mixture of 2-nitro-N-((tetrahydro-2H-pyran-4-
yl)methyl)aniline (1.5 g, 6.4 mmol), 1,5-dimethyl-6-oxo-1,6-dihy-
dropyridine-3-carbaldehyde (0.96 g, 6.4 mmol), and Na2S2O4 (2.0 g,
11.4 mmol) in EtOH (2 mL) and water (1 mL) were added in a
sealed tube and stirred at 100 °C for 16 h. The reaction mixture was
diluted with water (100 mL) and extracted with EtOAc (3 × 100
mL). The organic layer was washed with brine (250 mL), separated,
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude material was purified by column chromatography
1
ple gum (125 mg, 0.57 mmol, 100% yield). H NMR (400 MHz,
DMSO-d6): δ 6.53 (dd, J = 7.5, 1.5 Hz, 1 H), 6.47 (td, J = 7.5, 1.5 Hz,
1 H), 6.42 (dd, J = 7.5, 1.5 Hz, 1 H), 6.36 (td, J = 7.5, 1.5 Hz, 1 H),
4.46 (s, 2 H), 3.99 (d, J = 8.5 Hz, 1 H), 3.91−3.84 (m, 2 H), 3.29−
3.19 (m, 3 H), 1.74 (d, J = 13.0 Hz, 1 H), 1.71−1.61 (m, 1 H), 1.58
(d, J = 13.0 Hz, 1 H), 1.31 (td, J = 13.0, 4.5 Hz, 1 H), 1.25 (td, J =
13.0, 4.5 Hz, 1 H), 1.06 (d, J = 6.5 Hz, 3 H); LC−MS (method A) m/
z: 221 [(M + H)+]; Rt: 0.93 min; 91% purity. Step c (ii): batch 1:
racemic N1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)benzene-1,2-diamine
(60 mg, 0.27 mmol) and 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-
carbaldehyde (45 mg, 0.30 mmol) were dissolved in DMF (1.5 mL)
and water (0.05 mL). Potassium peroxymonosulfate (109 mg, 0.18
mmol) was added, and the reaction mixture was evacuated and
purged with nitrogen (×3) and left to stir for 18 h at rt under an
atmosphere of nitrogen. The reaction mixture was diluted with DCM
(30 mL) and washed with water (30 mL). The aqueous phase was
extracted with DCM (3 × 30 mL), and the organic layers were
S
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Journal of Medicinal Chemistry
Article
combined and dried over a hydrophobic frit. The solvent was
removed in vacuo, and the residue was dissolved in 1:1 DMSO/
MeOH (0.6 mL) and purified by MDAP (method B). The solvent
was evaporated in vacuo to give batch 1 racemic 1,3-dimethyl-5-(1-(1-
(tetrahydro-2H-pyran-4-yl)ethyl)-1H-benzo[d]imidazol-2-yl)pyridin-
2(1H)-one as a brown solid (66 mg). Batch 2: racemic N1-(1-
(tetrahydro-2H-pyran-4-yl)ethyl)benzene-1,2-diamine (65 mg, 0.30
mmol) and 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde
(45 mg, 0.30 mmol) were dissolved in DMF (1.5 mL) and water
(0.05 mL). Potassium peroxymonosulfate (118 mg, 0.19 mmol) was
added, and the reaction mixture was stirred for 5 h at rt under an
atmosphere of nitrogen. The reaction mixture was diluted with water
(30 mL). The aqueous phase was extracted with DCM (4 × 30 mL),
and the organic layers were combined and dried over a hydrophobic
frit. The solvent was removed in vacuo, and the residue was dissolved
in 1:1 DMSO/MeOH (0.6 mL) and purified by MDAP (method B).
The solvent was evaporated in vacuo to give batch 2 racemic 1,3-
dimethyl-5-(1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-benzo[d]-
imidazole-2-yl)pyridin-2(1H)-one as a brown solid (56 mg). The two
batches (batch 1, 36 mg and batch 2, 50 mg) were combined and
dissolved in EtOH (2 mL). Two injections were made onto a
Chiralpak AD-H column (250 mm × 30 mm). An isocratic system of
30% EtOH in heptane with a flow rate of 25 mL/min was used at rt.
The UV detection was performed at 215 nm. The appropriate
fractions were combined and evaporated in vacuo to give the title
compounds. Isomer 1 20b (28 mg, 0.08 mmol, 14% combined yield).
>99% ee determined by HPLC analysis on a Chiralpak AD-H (250
mm × 4.6 mm) column. Peak elution at Rt: 8.88 min. An isocratic
system of 30% EtOH in heptane with a flow rate of 1 mL/min was
MHz, CDCl3): δ 8.18 (dd, J = 8.5, 1.5 Hz, 1 H), 8.12 (d, J = 8.5 Hz, 1
H), 7.40 (ddd, J = 8.5, 7.0, 1.5 Hz, 1 H), 6.88 (d, J = 8.5 Hz, 1 H),
6.61 (ddd, J = 8.5, 7.0, 1.5 Hz, 1 H), 4.01 (dd, J = 11.5, 4.5 Hz, 2 H),
3.50−3.43 (m, 1 H), 3.42−3.34 (m, 2 H), 1.88−1.74 (m, 2 H), 1.74−
1.67 (m, 1 H), 1.65−1.58 (m, 1 H), 1.56−1.40 (m, 3 H), 0.96 (t, J =
7.5 Hz, 3 H); LC−MS (method A) m/z: 263 [(M + H)+]; Rt: 1.24
min; 99% purity. Step b (i): 2-Nitro-N-(1-(tetrahydro-2H-pyran-4-
yl)propyl)aniline (265 mg, 1.00 mmol), Na2S2O4 (524 mg, 3.01
mmol), and 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde
(212 mg, 1.40 mmol) were suspended in EtOH (1.5 mL) and water
(0.75 mL) within a sealed vessel and heated in a microwave at 100 °C
for 65 min. The reaction mixture was allowed to cool to rt. The
reaction mixture was diluted with saturated aqueous NaHCO3 (30
mL). The aqueous phase was extracted with DCM (4 × 30 mL), and
the organic extracts were combined and dried over a hydrophobic frit.
The solvent was removed in vacuo, and the residue was taken up into
DCM (5 mL) and purified by normal phase column chromatography
on a 40 g silica cartridge eluting with 0−100% EtOAc in cyclohexane
over 10 column volumes followed by 100% EtOAc over 10 column
volumes and MeOH over 10 column volumes. The desired fractions
were combined, and the solvent was removed in vacuo to give racemic
1,3-dimethyl-5-(1-(1-(tetrahydro-2H-pyran-4-yl)propyl)-1H-benzo-
[d]imidazol-2-yl)pyridin-2(1H)-one (330 mg). Racemate (324 mg)
was dissolved in EtOH (3 mL). Three injections were made onto a
Chiralcel OD-H column (250 mm × 30 mm). An isocratic system of
10% EtOH in heptane with a flow rate of 30 mL/min was used at rt.
The UV detection was performed at 215 nm. The appropriate
fractions were combined and evaporated in vacuo. Mixed fractions
were reprocessed using the same conditions to give the title
compounds. Isomer 1 20c (170 mg, 0.47 mmol, 47% yield). >99%
ee determined by HPLC analysis on a Chiralcel OD-H (250 mm ×
4.6 mm) column. Peak elution at Rt: 26.02 min. An isocratic system of
10% EtOH in heptane with a flow rate of 1 mL/min was used at rt.
1
used at rt. The UV detection was performed at 215 nm; H NMR
(400 MHz, CDCl3): δ 7.78 (dd, J = 7.0, 2.0 Hz, 1 H), 7.66 (d, J = 2.0
Hz, 1 H), 7.57 (dd, J = 7.0, 2.0 Hz, 1 H), 7.36−7.33 (m, 1 H), 7.31
(td, J = 7.0, 2.0 Hz, 1 H), 7.27 (td, J = 7.0, 2.0 Hz, 1 H), 4.15 (dq, J =
10.5, 7.0 Hz, 1 H), 4.03 (dd, J = 11.5, 4.0 Hz, 1 H), 3.75 (dd, J = 11.5,
4.0 Hz, 1 H), 3.66 (s, 3 H), 3.39 (td, J = 11.5, 2.0 Hz, 1 H), 3.13 (td, J
= 11.5, 2.0 Hz, 1 H), 2.41 (tdt, J = 11.5, 10.5, 4.0 Hz, 1 H), 2.25 (s, 3
H), 1.85 (ddd, J = 13.0, 4.0, 2.0 Hz, 1 H), 1.75 (d, J = 7.0 Hz, 3 H),
1.35 (dtd, J = 13.0, 11.5, 4.0 Hz, 1 H), 0.98 (dtd, J = 13.0, 11.5, 4.0
Hz, 1 H), 0.70 (ddd, J = 13.0, 4.0, 2.0 Hz, 1 H); LC−MS (method A)
m/z: 352 [(M + H)+]; Rt: 0.87 min; 100% purity. Isomer 2 20b′ (28
mg, 0.08 mmol, 14% combined yield). 99% ee determined by HPLC
analysis on a Chiralpak AD-H (250 mm × 4.6 mm) column. Peak
elution at Rt: 10.83 min. An isocratic system of 30% EtOH in heptane
with a flow rate of 1 mL/min was used at rt. The UV detection was
performed at 215 nm; 1H NMR (400 MHz, CDCl3): δ 7.78 (dd, J =
7.0, 2.0 Hz, 1 H), 7.66 (d, J = 2.0 Hz, 1 H), 7.57 (dd, J = 7.0, 2.0 Hz,
1 H), 7.36−7.33 (m, 1 H), 7.31 (td, J = 7.0, 2.0 Hz, 1 H), 7.28 (td, J =
7.0, 2.0 Hz, 2 H), 4.15 (dq, J = 10.5, 7.0 Hz, 1 H), 4.03 (dd, J = 11.5,
4.0 Hz, 1 H), 3.75 (dd, J = 11.5, 4.0 Hz, 1 H), 3.66 (s, 3 H), 3.39 (td,
J = 11.5, 2.0 Hz, 1 H), 3.13 (td, J = 11.5, 2.0 Hz, 1 H), 2.41 (tdt, J =
11.5, 10.5, 4.0 Hz, 1 H), 2.25 (s, 3 H), 1.85 (ddd, J = 13.0, 4.0, 2.0
Hz, 1 H), 1.75 (d, J = 7.0 Hz, 3 H), 1.35 (dtd, J = 13.0, 11.5, 4.0 Hz, 2
H), 0.98 (dtd, J = 13.0, 11.5, 4.0 Hz, 1 H), 0.70 (ddd, J = 13.0, 4.0, 2.0
Hz, 1 H); LC−MS (method A) m/z: 352 [(M + H)+]; Rt: 0.87 min;
100% purity.
1
The UV detection was performed at 215 nm; H NMR (400 MHz,
CDCl3): δ 7.79 (dd, J = 7.5, 1.5 Hz, 1 H), 7.61 (d, J = 1.5 Hz, 1 H),
7.56 (d, J = 7.5 Hz, 1 H), 7.43 (s, 1 H), 7.31 (td, J = 7.5, 1.5 Hz, 1 H),
7.29−7.25 (m, 1 H), 4.05 (dd, J = 11.5, 4.0 Hz, 1 H), 3.97 (td, J =
11.0, 4.0 Hz, 1 H), 3.82 (dd, J = 11.5, 4.0 Hz, 1 H), 3.65 (s, 3 H),
3.41 (td, J = 11.5, 2.0 Hz, 1 H), 3.19 (td, J = 11.5, 2.0 Hz, 1 H), 2.48−
2.34 (m, 1 H), 2.25 (s, 3 H), 2.29−2.16 (m, 1 H), 2.13−2.02 (m, 1
H), 1.87 (ddd, J = 13.0, 4.0, 2.0 Hz, 1 H), 1.39 (dtd, J = 13.0, 11.5, 4.0
Hz, 1 H), 1.14 (dtd, J = 13.0, 11.5, 4.0 Hz, 1 H), 0.93−0.86 (m, 1 H),
0.77 (t, J = 7.5 Hz, 3 H); LC−MS (method F) m/z: 366 [(M + H)+];
Rt: 0.66 min; 100% purity. Isomer 2 20c′ (151 mg, 0.41 mmol, 41%
yield). 99% ee determined by HPLC analysis on a Chiralcel OD-H
(250 mm × 4.6 mm) column. Peak elution at Rt: 30.83 min. An
isocratic system of 10% EtOH in heptane with a flow rate of 1 mL/
1
min was used at rt. The UV detection was performed at 215 nm; H
NMR (400 MHz, CDCl3): δ 7.79 (dd, J = 7.5, 1.5 Hz, 1 H), 7.61 (d, J
= 1.5 Hz, 1 H), 7.56 (d, J = 7.5 Hz, 1 H), 7.43 (s, 1 H), 7.29−7.25
(m, 1 H), 7.31 (td, J = 7.5, 1.5 Hz, 1 H), 4.05 (dd, J = 11.5, 4.0 Hz, 1
H), 3.97 (td, J = 11.0, 4.0 Hz, 1 H), 3.82 (dd, J = 11.5, 4.0 Hz, 1 H),
3.65 (s, 3 H), 3.41 (td, J = 11.5, 2.0 Hz, 1 H), 3.19 (td, J = 11.5, 2.0
Hz, 1 H), 2.48−2.34 (m, 1 H), 2.25 (s, 3 H), 2.29−2.16 (m, 1 H),
2.13−2.02 (m, 1 H), 1.87 (ddd, J = 13.0, 4.0, 2.0 Hz, 1 H), 1.39 (dtd,
J = 13.0, 11.5, 4.0 Hz, 1 H), 1.14 (dtd, J = 13.0, 11.5, 4.0 Hz, 1 H),
0.93−0.86 (m, 1 H), 0.77 (t, J = 7.5 Hz, 3 H); LC−MS (method F)
m/z: 366 [(M + H)+]; Rt: 0.66 min; 100% purity.
1,3-Dimethyl-5-(1-((tetrahydro-2H-pyran-3-yl)methyl)-1H-
benzo[d]imidazol-2-yl)pyridin-2(1H)-one (20d and 20d′). Step a: 1-
fluoro-2-nitrobenzene (0.11 mL, 1.042 mmol), DIPEA (0.40 mL,
2.292 mmol), and (tetrahydro-2H-pyran-3-yl)methanamine (144 mg,
1.25 mmol) were suspended in NMP (1 mL) within a sealed vial and
heated in a microwave at 125 °C for 1 h. The reaction mixture was
allowed to reach rt and then diluted with water (40 mL) and extracted
with EtOAc (5 × 40 mL). The organic layers were combined and
dried over a hydrophobic frit, and the solvent was removed in vacuo.
The residue was taken up into DCM (5 mL) and purified by normal
phase column chromatography on a 40 g silica column and eluted
with 0−100% EtOAc in cyclohexane over 10 column volumes. The
1,3-Dimethyl-5-(1-(1-(tetrahydro-2H-pyran-4-yl)propyl)-1H-
benzo[d]imidazole-2-yl)pyridin-2(1H)-one (20c and 20c′). Step a:
1-fluoro-2-nitrobenzene (0.11 mL, 1.04 mmol), DIPEA (0.22 mL,
1.26 mmol), and 1-(tetrahydro-2H-pyran-4-yl)propan-1-amine (179
mg, 1.25 mmol) were suspended in NMP (1 mL) within a sealed vial
and heated in a microwave at 125 °C for 1 h. The mixture was
allowed to reach rt and then diluted with water (40 mL) and extracted
with EtOAc (5 × 40 mL). The organic layers were combined and
dried over a hydrophobic frit, and the solvent was removed in vacuo.
The residue was taken up into DCM (5 mL) and purified by normal
phase column chromatography on a 40 g silica column and eluting
with 0−100% EtOAc in cyclohexane over 10 column volumes. The
desired fractions were combined, and the solvent was removed in
vacuo to give 2-nitro-N-(1-(tetrahydro-2H-pyran-4-yl)propyl)aniline
1
as an orange gum (265 mg, 1.00 mmol, 96% yield). H NMR (400
T
DOI: 10.1021/acs.jmedchem.9b01670
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
desired fractions were combined, the solvent was removed in vacuo,
and the residue was dried in a vacuum oven overnight to give 2-nitro-
N-((tetrahydro-2H-pyran-3-yl)methyl)aniline as an orange gum (201
mg, 0.85 mmol, 82% yield). 1H NMR (400 MHz, CDCl3): δ 8.19 (dd,
J = 8.5, 1.5 Hz, 1 H), 8.15−8.05 (m, 1 H), 7.45 (ddd, J = 8.5, 7.0, 1.5
Hz, 1 H), 6.85 (dd, J = 8.5, 1.5 Hz, 1 H), 6.66 (ddd, J = 8.5, 7.0, 1.5
Hz, 1 H), 3.96 (ddd, J = 11.0, 4.0, 1.5 Hz, 1 H), 3.86 (dt, J = 11.0, 4.0
Hz, 1 H), 3.51 (ddd, J = 11.0, 9.0, 4.0 Hz, 1 H), 3.34 (dd, J = 11.0, 9.0
Hz, 1 H), 3.31−3.17 (m, 2 H), 2.10−1.95 (m, 2 H), 1.76−1.59 (m, 2
H), 1.48−1.37 (m, 1 H); LC−MS (method A): no mass ion
observed; Rt: 1.12 min; 98% purity. Step b (i): 2-nitro-N-
((tetrahydro-2H-pyran-3-yl)methyl)aniline (201 mg, 0.85 mmol),
Na2S2O4 (444 mg, 2.55 mmol), and 1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carbaldehyde (180 mg, 1.19 mmol) were sus-
pended in EtOH (1.5 mL) and water (0.75 mL) within a sealed vessel
and heated in a microwave at 100 °C for 65 min. The reaction
mixture was allowed to cool to rt. The reaction mixture was diluted
with water (30 mL). The aqueous phase was extracted with DCM (4
× 30 mL), and the organic layers were combined and dried over a
hydrophobic frit. The solvent was removed in vacuo, and the residue
was dissolved in 1:1 DMSO/MeOH (0.6 mL) and purified by MDAP
(method B). The solvent was evaporated in vacuo to give racemic 1,3-
dimethyl-5-(1-((tetrahydro-2H-pyran-3-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)pyridin-2(1H)-one (148 mg). Racemate (144 mg) was
dissolved in EtOH (2 mL). Two injections were made onto a
Chiralpak AD-H column (250 mm × 30 mm). An isocratic system of
40% EtOH (containing 0.2% isopropylamine) in heptane (containing
0.2% isopropylamine) with a flow rate of 30 mL/min was used at rt.
The UV detection was performed at 215 nm. The appropriate
fractions were combined and evaporated in vacuo to give the title
compounds. Isomer 1 20d (68 mg, 0.20 mmol, 24% yield). >99% ee
determined by HPLC analysis on a Chiralpak AD-H (250 mm × 4.6
mm) column. Peak elution at Rt: 11.23 min. An isocratic system of
50% EtOH (containing 0.2% isopropylamine) in heptane with a flow
rate of 1 mL/min was used at rt. The UV detection was performed at
removed under a stream of nitrogen, and the residue was purified by
MDAP (method B). Step b (ii): to the dried material were added
DCM (0.2 mL) and TFA (0.2 mL), and the solution was left for 2 h.
The solvent was removed under a stream of nitrogen, and the residue
was purified by MDAP (method B) to give the title compound (6 mg,
1
0.02 mmol, 2% overall yield). H NMR (400 MHz, DMSO-d6): δ
8.11 (d, J = 2.5 Hz, 1 H), 7.74−7.71 (m, 1 H), 7.62 (dd, J = 7.5, 1.5
Hz, 1 H), 7.61 (dd, J = 7.0, 1.5 Hz, 1 H), 7.25 (ddd, J = 7.5, 7.0, 1.5
Hz, 1 H), 7.21 (ddd, J = 7.5, 7.0, 1.5 Hz, 1 H), 4.39 (d, J = 4.5 Hz, 1
H), 4.19 (d, J = 7.5 Hz, 2 H), 3.56 (s, 3 H), 3.29−3.21 (m, 1 H), 2.10
(s, 3 H), 1.75−1.58 (m, 3 H), 1.38−1.28 (m, 2 H), 1.00−0.85 (m, 4
H); LC−MS (method F) m/z: 352 [(M + H)+]; Rt: 0.47 min; 96%
purity.
1,3-Dimethyl-5-(1-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-
benzo[d]imidazole-2-yl)pyridin-2(1H)-one (20f). Step a: to a
solution of 1-fluoro-2-nitrobenzene (50 mg, 0.35 mmol) in 2-
MeTHF (1 mL) were added 2-(tetrahydro-2H-pyran-4-yl)-
ethanamine (55 mg, 0.43 mmol) and DIPEA (0.19 mL, 1.06
mmol). The reaction vessel was sealed and heated in a microwave at
110 °C for 20 min. After cooling the reaction, further 2-(tetrahydro-
2H-pyran-4-yl)ethanamine (55 mg, 0.43 mmol) was added and the
vial was capped and heated to 120 °C for 20 min. The solvent was
removed under a stream of nitrogen. Step b (i): to the dried material
were added 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde
(58.9 mg, 0.39 mmol) and Na2S2O4 (185 mg, 1.06 mmol) as a
solution in EtOH (1.0 mL), followed by water (0.5 mL). The sealed
vessel was heated to 100 °C for 18 h. The solvent was removed under
a stream of nitrogen. The reaction mixture was partitioned between
saturated aqueous NaHCO3 (3 mL) and DCM (2 × 3 mL). The
DCM was removed under a stream of nitrogen. The sample was
dissolved in DMSO (1 mL) and purified by MDAP (method B). The
solvent was evaporated under a stream of nitrogen to give the title
1
compound (12 mg, 0.03 mmol, 9% overall yield). H NMR (600
MHz, DMSO-d6): δ 8.11 (d, J = 2.5 Hz, 1 H), 7.73−7.71 (m, 1 H),
7.64−7.59 (m, 2 H), 7.27 (t, J = 7.5 Hz, 1 H), 7.22 (t, J = 7.5 Hz, 1
H), 4.32 (t, J = 7.5 Hz, 2 H), 3.77 (dd, J = 11.5, 3.0 Hz, 2 H), 3.56 (s,
3 H), 3.17 (td, J = 11.5, 2.0 Hz, 2 H), 2.11 (s, 3 H), 1.65 (q, J = 7.5
Hz, 2 H), 1.47 (dd, J = 11.5, 2.0 Hz, 2 H), 1.44−1.36 (m, 1 H), 1.16
(qd, J = 11.5, 4.5 Hz, 2 H); LC−MS (method F) m/z: 352 [(M +
H)+]; Rt: 0.55 min; 100% purity.
1
215 nm; H NMR (400 MHz, CDCl3): δ 7.81 (d, J = 2.0 Hz, 1 H),
7.79−7.76 (m, 1 H), 7.58−7.56 (m, 1 H), 7.42−7.37 (m, 1 H), 7.34−
7.29 (m, 2 H), 4.28 (dd, J = 14.5, 8.5 Hz, 1 H), 4.13 (dd, J = 14.5, 7.0
Hz, 1 H), 3.76−3.69 (m, 1 H), 3.66 (s, 3 H), 3.62−3.48 (m, 2 H),
3.25 (dd, J = 11.5, 7.5 Hz, 1 H), 2.26 (s, 3 H), 2.25−2.18 (m, 1 H),
1.76−1.67 (m, 1 H), 1.67−1.59 (m, 1 H), 1.55−1.45 (m, 1 H), 1.37−
1.27 (m, 1 H); LC−MS (method A) m/z: 338 [(M + H)+]; Rt: 0.85
min; 100% purity. Isomer 2 20d′ (74 mg, 0.22 mmol, 26% yield).
>99% ee determined by HPLC analysis on a Chiralpak AD-H (250
mm × 4.6 mm) column. Peak elution at Rt: 17.57 min. An isocratic
system of 50% EtOH (containing 0.2% isopropylamine) in heptane
with a flow rate of 1 mL/min was used at rt. The UV detection was
5-(1-(2-Oxaspiro[3.3]heptan-6-ylmethyl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one (20g). Step a: to a solution of 1-
fluoro-2-nitrobenzene (100 mg, 0.71 mmol) in 2-MeTHF (1 mL)
were added 2-oxaspiro[3.3]heptan-6-ylmethanamine (108 mg, 0.85
mmol) and DIPEA (0.37 mL, 2.13 mmol). The reaction vessel was
sealed and heated in a microwave at 110 °C for 30 min. The solvent
was removed under a stream of nitrogen. The residue was purified by
MDAP (method B). Step b (i): to the dried material were added 1,5-
dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde (118 mg, 0.78
mmol) and Na2S2O4 (370 mg, 2.13 mmol) as a solution in EtOH
(1.0 mL), followed by water (0.5 mL). The sealed vessel was heated
to 100 °C for 4 h. The solvent was removed under a stream of
nitrogen. The reaction mixture was partitioned between saturated
aqueous NaHCO3 (1.5 mL) and DCM (2 × 3 mL). The DCM was
removed under a stream of nitrogen. The residue was dissolved in
DMSO (1 mL) and purified by MDAP (method B) to give the title
1
performed at 215 nm; H NMR (400 MHz, CDCl3): δ 7.81 (d, J =
2.0 Hz, 1 H), 7.79−7.76 (m, 1 H), 7.58−7.56 (m, 1 H), 7.42−7.37
(m, 1 H), 7.34−7.28 (m, 2 H), 4.28 (dd, J = 14.5, 8.5 Hz, 1 H), 4.13
(dd, J = 14.5, 7.0 Hz, 1 H), 3.76−3.69 (m, 1 H), 3.66 (s, 3 H), 3.62−
3.48 (m, 2 H), 3.25 (dd, J = 11.5, 7.5 Hz, 1 H), 2.26 (s, 3 H), 2.25−
2.18 (m, 1 H), 1.76−1.67 (m, 1 H), 1.67−1.59 (m, 1 H), 1.55−1.45
(m, 1 H), 1.37−1.27 (m, 1 H); LC−MS (method A) m/z: 338 [(M +
H)+]; Rt: 0.85 min; 100% purity.
5-(1-((trans-4-Hydroxycyclohexyl)methyl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one (20e). Step a: to a solution of 1-
fluoro-2-nitrobenzene (100 mg, 0.71 mmol) in 2-methyl tetrahy-
drofuran (2-MeTHF, 1 mL) were added (trans-4-(tert-butoxy)-
cyclohexyl)methanamine (158 mg, 0.85 mmol) and DIPEA (0.37
mL, 2.13 mmol). The reaction vessel was sealed and heated in a
microwave at 110 °C for 20 min. The reaction was cooled and then
heated to 110 °C for 20 min. The solvent was removed under a
stream of nitrogen. Step b (i): to the dried material were added 1,5-
dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde (118 mg, 0.78
mmol) and Na2S2O4 (370 mg, 2.13 mmol) as a solution in EtOH
(1.0 mL), followed by water (0.5 mL). The sealed vessel was heated
at 100 °C for 6 h. The solvent was removed under a stream of
nitrogen. The reaction mixture was partitioned between saturated
aqueous NaHCO3 (3 mL) and DCM (2 × 3 mL). The DCM was
1
compound (10 mg, 0.03 mmol, 4% overall yield). H NMR (600
MHz, DMSO-d6): δ 8.11 (s, 1 H), 7.72 (s, 1 H), 7.63 (d, J = 7.5 Hz, 1
H), 7.60 (d, J = 7.5 Hz, 1 H), 7.25 (t, J = 7.5 Hz, 1 H), 7.21 (t, J = 7.5
Hz, 1 H), 4.44 (s, 2 H), 4.34 (d, J = 7.5 Hz, 2 H), 4.32 (s, 2 H), 3.56
(s, 3 H), 2.41 (tt, J = 7.5 Hz, 1 H), 2.11 (s, 3 H), 2.09 (dd, J = 12.0,
7.5 Hz, 2 H), 1.81 (dd, J = 12.0, 7.5 Hz, 2 H); LC−MS (method F)
m/z: 350 [(M + H)+]; Rt: 0.56 min; 99% purity.
5-(1-(1-Methoxypropan-2-yl)-1H-benzo[d]imidazol-2-yl)-1,3-di-
methylpyridin-2(1H)-one (20h). Step a: to a solution of 1-fluoro-2-
nitrobenzene (100 mg, 0.71 mmol) in 2-MeTHF (1 mL) were added
1-methoxypropan-2-amine (1.42 mmol, 126 mg) and DIPEA (0.37
mL, 2.13 mmol). The reaction vessel was sealed and heated in a
microwave at 110 °C for 20 min. The reaction was cooled and then
heated to 120 °C for 20 min. The solvent was removed under a
U
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
Article
stream of nitrogen. Step b (i): to the dried material were added 1,5-
dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde (118 mg, 0.78
mmol) and Na2S2O4 (370 mg, 2.13 mmol) as a solution in EtOH
(1.0 mL), followed by water (0.5 mL). The sealed vessel was heated
to 100 °C for 18 h. The solvent was removed under a stream of
nitrogen. The sample was dissolved in DMSO (1 mL) and purified by
MDAP (method B) to give the title compound (66 mg, 0.21 mmol,
yl)methanamine (5.47 mL, 44.7 mmol) and DIPEA (11.7 mL, 67.1
mmol) were added. The reaction mixture was heated at 80 °C for 2 h
and then added to water (200 mL) and extracted with EtOAc (2 ×
200 mL). The combined organics were washed with water (200 mL)
and brine (200 mL), dried, and evaporated in vacuo to give the title
compound as a yellow solid (12.2 g, 38.7 mmol, 87% yield). 1H NMR
(400 MHz, CDCl3): δ 8.14 (t, J = 5.5 Hz, 1 H), 8.04 (d, J = 9.0 Hz, 1
H), 7.02 (d, J = 2.0 Hz, 1 H), 6.77 (dd, J = 9.0, 2.0 Hz, 1 H), 4.04
(dd, J = 11.0, 4.5 Hz, 2 H), 3.44 (td, J = 11.5, 2.0 Hz, 2 H), 3.20 (dd, J
= 6.5, 5.5 Hz, 2 H), 1.96 (ttt, J = 11.5, 6.5, 3.5 Hz, 1 H), 1.76 (ddd, J
= 13.0, 3.5, 2.0 Hz, 2 H), 1.44 (dtd, J = 13.0, 11.5, 4.5 Hz, 2 H). LC−
MS (method F) m/z: 315, 317 [(M + H)+]; Rt: 1.25 min; 100%
purity.
1
27% overall yield). H NMR (400 MHz, DMSO-d6): δ 8.02 (d, J =
2.5 Hz, 1 H), 7.79−7.74 (m, 1 H), 7.65−7.60 (m, 2 H), 7.24−7.19
(m, 2 H), 4.84−4.73 (m, 1 H), 4.02 (t, J = 10.0 Hz, 1 H), 3.68 (dd, J
= 10.0, 4.5 Hz, 1 H), 3.55 (s, 3 H), 3.15 (s, 3 H), 2.10 (s, 3 H), 1.54
(d, J = 7.0 Hz, 3 H); LC−MS (method F) m/z: 312 [(M + H)+]; Rt:
0.53 min; 99% purity.
5-(1-(1,3-Dimethoxypropan-2-yl)-1H-benzo[d]imidazole-2-yl)-
1,3-dimethylpyridin-2(1H)-one (20i). Step a: a mixture of 1-fluoro-2-
nitrobenzene (0.15 mL, 1.42 mmol), DIPEA (0.37 mL, 2.12 mmol),
and 1,3-dimethoxypropan-2-amine (340 mg, 2.85 mmol) in a
microwave vial was diluted with anhydrous NMP (2 mL). The vial
was sealed and heated in a microwave at 125 °C for 30 min. The
reaction mixture was diluted with water (5 mL) and extracted with
EtOAc (3 × 5 mL). The organic extracts were combined, washed with
brine (15 mL), and dried through a hydrophobic frit. The solvent was
removed in vacuo, and the residue was purified by MDAP (method
B). The appropriate fractions were combined, and the solvent was
removed by rotary evaporation. The remaining mixture was diluted
with water (3 mL) and extracted with EtOAc (3 × 3 mL). The
organic extracts were combined and dried through a hydrophobic frit
to give N-(1,3-dimethoxypropan-2-yl)-2-nitroaniline as an orange oil
5-(5-Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22a). 4-Bromo-2-
nitro-N-((tetrahydro-2H-pyran-4-yl)methyl)aniline 21a (15.6 g, 49.5
mmol) and 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde
(7.48 g, 49.5 mmol) were combined in EtOH (375 mL) and water
(150 mL). Na2S2O4 (25.9 g, 148 mmol) was added and the mixture
heated to 90 °C. The reaction was cooled and concentrated to remove
the EtOH. The residue was partitioned between 10:1 DCM/iPrOH
(500 mL) and water (400 mL). The aqueous layer was re-extracted
with 10:1 DCM/iPrOH (500 mL), and the combined organics were
dried with Na2SO4, filtered, and concentrated in vacuo to yield an
orange oil. The crude product was applied to a 340 g silica cartridge in
the minimum amount of DCM and eluted with 5% (3:1 EtOAc/
EtOH) in cyclohexane for 2 column volumes, then 5−50% (3:1
EtOAc/EtOH) over 10 column volumes, and then held at 50% for 15
column volumes. The appropriate fractions were concentrated in
vacuo to give the title compound as white foam (9.21 g, 21.0 mmol,
43% yield). 1H NMR (400 MHz, CDCl3): δ 7.87 (d, J = 1.5 Hz, 1 H),
7.73 (d, J = 2.5 Hz, 1 H), 7.48−7.45 (m, 1 H), 7.39 (dd, J = 8.5, 1.5
Hz, 1 H), 7.24 (d, J = 8.5 Hz, 1 H), 4.11 (d, J = 7.5 Hz, 2 H), 3.88
(dd, J = 11.5, 3.5 Hz, 2 H), 3.64 (s, 3 H), 3.22 (td, J = 11.5, 2.0 Hz, 2
H), 2.23 (s, 3 H), 2.11−1.95 (m, 1 H), 1.35−1.18 (m, 4 H); LC−MS
(method A) m/z: 416, 418 [(M + H)+]; Rt: 0.96 min; 100% purity.
5-(6-Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22b). 5-Bromo-2-
nitro-N-((tetrahydro-2H-pyran-4-yl)methyl)aniline 21b (12.2 g, 38.7
mmol) and 1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde
(6.4 g, 42.6 mmol) were combined in EtOH (200 mL) and water
(100 mL), and the mixture was heated to 80 °C; then, Na2S2O4 (20.2
g, 116 mmol) was added, and the resulting suspension was heated for
18 h at 80 °C. The mixture was evaporated in vacuo to approximately
half its original volume and then extracted with EtOAc (3 × 100 mL),
and the combined organics were dried and evaporated in vacuo to
give colorless foam. This was dissolved in DCM (30 mL) and loaded
onto a 340 g silica column and then eluted with 0−25% EtOH in
EtOAc, and product-containing fractions evaporated in vacuo to give
the title compound as a pale yellow solid (10.2 g, 24.5 mmol, 63%
yield). 1H NMR (400 MHz, CDCl3): δ 7.73 (d, J = 2.5 Hz, 1 H), 7.62
(d, J = 8.5 Hz, 1 H), 7.54 (d, J = 1.5 Hz, 1 H), 7.49−7.46 (m, 1 H),
7.41 (dd, J = 8.5, 1.5 Hz, 1 H), 4.11 (d, J = 7.5 Hz, 2 H), 3.91 (dd, J =
11.5, 3.5 Hz, 2 H), 3.66 (s, 3 H), 3.27 (td, J = 11.5, 2.5 Hz, 2 H), 2.25
(s, 3 H), 2.15−2.04 (m, 1 H), 1.38−1.21 (m, 4 H); LC−MS (method
F) m/z: 416, 418 [(M + H)+]; Rt: 0.85 min; 100% purity.
1
(275 mg, 1.15 mmol, 80% yield). H NMR (400 MHz, CDCl3): δ
8.34 (d, J = 7.0 Hz, 1 H), 8.18 (dd, J = 8.5, 1.5 Hz, 1 H), 7.43 (ddd, J
= 8.5, 7.0, 1.5 Hz, 1 H), 6.96 (dd, J = 8.5, 1.0 Hz, 1 H), 6.65 (ddd, J =
8.5, 7.0, 1.0 Hz, 1 H), 3.94−3.84 (m, 1 H), 3.65−3.53 (m, 4 H), 3.42
(s, 6 H); LC−MS (method F) m/z: 241 [(M + H)+]; Rt: 1.07 min;
100% purity. Step b (i): to a mixture of 1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carbaldehyde (185 mg, 1.22 mmol) and Na2S2O4
(580 mg, 3.33 mmol) in a microwave vessel was added a solution of
N-(1,3-dimethoxypropan-2-yl)-2-nitroaniline (267 mg, 1.11 mmol) in
EtOH (2 mL) followed by water (1 mL). The vial was sealed and
heated in a microwave at 100 °C for 65 min. The reaction mixture
was diluted with water (3 mL) and extracted with EtOAc (2 × 5 mL).
The organic extracts were combined and passed through a
hydrophobic frit, and the solvent was removed under vacuum. The
residue was purified by MDAP (method B). The appropriate fractions
were combined, and the solvent was removed by rotary evaporation to
give the title compound as a white solid (129 mg, 0.38 mmol, 34%
yield). 1H NMR (400 MHz, DMSO-d6): δ 8.05 (d, J = 2.5 Hz, 1 H),
7.82−7.77 (m, 1 H), 7.70−7.67 (m, 1 H), 7.65−7.60 (m, 1 H), 7.25−
7.17 (m, 2 H), 4.84 (tt, J = 9.0, 4.5 Hz, 1 H), 4.02 (dd, J = 10.5, 9.0
Hz, 2 H), 3.77 (dd, J = 10.5, 4.5 Hz, 2 H), 3.54 (s, 3 H), 3.16 (s, 6
H), 2.09 (s, 3 H); LC−MS (method F) m/z: 342 [(M + H)+]; Rt:
0.60 min; 100% purity.
4-Bromo-2-nitro-N-((tetrahydro-2H-pyran-4-yl)methyl)aniline
(21a). 4-Bromo-1-fluoro-2-nitrobenzene (5 g, 22.7 mmol) was taken
up in 2-MeTHF (50 mL). DIPEA (6.0 mL, 34.1 mmol) and
(tetrahydro-2H-pyran-4-yl)methanamine (3.93 g, 34.1 mmol) were
added, and the reaction mixture was heated to 80 °C. The reaction
was cooled and diluted with EtOAc (100 mL) and then washed with
water (100 mL) and brine (100 mL). The organic layer was dried
with Na2SO4, filtered, and concentrated in vacuo to yield the title
5-(5-(Dimethylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22c). A
mixture of 5-(5-bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one 22a (100
mg, 0.24 mmol), 2 M dimethylamine in THF (0.6 mL, 1.20
mmol), Pd2(dba)3 (9 mg, 0.01 mmol), JohnPhos (9 mg, 0.03 mmol),
and NaOtBu (35 mg, 0.36 mmol) in toluene (2 mL) in a sealed
microwave vial was purged with nitrogen and heated in a microwave
at 90 °C for 20 min. The reaction mixture was filtered over Celite, and
the pad was washed with EtOAc (20 mL). The solvent was removed
in vacuo, and the residue was dissolved in 1:1 DMSO/MeOH (2 mL)
and purified by MDAP (method A). The solvent was evaporated in
vacuo, and the residue was dissolved in MeOH (0.5 mL). The
solution was applied to a 0.5 g MeOH-preconditioned aminopropyl
1
compound as an orange solid (7.15 g, 21.6 mmol, 95% yield). H
NMR (400 MHz, CDCl3): δ 8.33 (d, J = 2.5 Hz, 1 H), 8.13 (t, J = 5.5
Hz, 1 H), 7.51 (dd, J = 9.0, 2.5 Hz, 1 H), 6.77 (d, J = 9.0 Hz, 1 H),
4.03 (dd, J = 11.5, 4.5 Hz, 2 H), 3.42 (td, J = 11.5, 2.0 Hz, 2 H), 3.21
(dd, J = 6.5, 5.5 Hz, 2 H), 1.95 (ttt, J = 11.5, 6.5, 3.5 Hz, 1 H), 1.75
(ddd, J = 13.0, 3.5, 2.0 Hz, 2 H), 1.43 (dtd, J = 13.0, 11.5, 4.5 Hz, 2
H); LC−MS (method A) m/z: not observed [(M + H)+]; Rt: 1.25
min; 90% purity.
5-Bromo-2-nitro-N-((tetrahydro-2H-pyran-4-yl)methyl)aniline
(21b). 4-Bromo-2-fluoro-1-nitrobenzene (10 g, 44.7 mmol) was
dissolved in 2-MeTHF (100 mL), and (tetrahydro-2H-pyran-4-
V
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
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cartridge which was then washed with MeOH (3 mL). The wash was
evaporated under a stream of nitrogen, and the residue was dried in a
high-vacuum oven to give the title compound as an off-white solid (48
mg, 0.13 mmol, 53% yield). 1H NMR (400 MHz, CDCl3): δ 7.72 (d,
J = 2.5 Hz, 1 H), 7.52−7.48 (m, 1 H), 7.25 (d, J = 9.0 Hz, 1 H), 7.11
(d, J = 2.5 Hz, 1 H), 6.92 (dd, J = 9.0, 2.5 Hz, 1 H), 4.08 (d, J = 7.5
Hz, 2 H), 3.90 (dd, J = 11.5, 4.0 Hz, 2 H), 3.65 (s, 3 H), 3.24 (td, J =
11.5, 2.0 Hz, 2 H), 2.99 (s, 6 H), 2.25 (s, 3 H), 2.09 (ttt, J = 11.5, 7.5,
3.5 Hz, 1 H), 1.36 (ddd, J = 13.0, 3.5, 2.0 Hz, 2 H), 1.27 (dtd, J =
13.0, 11.5, 4.0 Hz, 2 H); LC−MS (method F) m/z: 381 [(M + H)+];
Rt: 0.48 min; 100% purity.
1.11 (m, 2 H). LC−MS (method F) m/z: 368 [(M + H)+]; Rt: 0.53
min; 96% purity.
1,3-Dimethyl-5-(5-methyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-
1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (22g). 11 mg, 0.03
1
mmol, 7% yield. H NMR (600 MHz, DMSO-d6): δ 8.11 (s, 1 H),
7.72 (s, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.40 (s, 1 H), 7.08 (d, J = 8.0
Hz, 1 H), 4.24 (d, J = 7.5 Hz, 2 H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H),
3.55 (s, 3 H), 3.10 (t, J = 11.5 Hz, 2 H), 2.41 (s, 3 H), 2.10 (s, 3 H),
1.94 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.19 (dd, J = 11.5 Hz, 2 H), 1.13
(qd, J = 11.5, 3.5 Hz, 2 H). LC−MS (method F) m/z: 352 [(M +
H)+]; Rt: 0.54 min; 100% purity.
1,3-Dimethyl-5-(6-methyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-
5-(6-(Dimethylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22d). 5-(6-
Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one 22b (50 mg, 0.12 mmol),
JohnPhos (4.6 mg, 0.015 mmol), 2 M dimethylamine in THF (0.10
mL, 0.20 mmol), NaOtBu (23.1 mg, 0.24 mmol), and Pd2(dba)3 (4.5
mg, 0.05 mmol) were suspended in 1,4-dioxane (1 mL). The reaction
vessel was sealed and heated in a microwave at 90 °C for 20 min. The
reaction mixture was allowed to cool to rt, and JohnPhos (4.6 mg,
0.015 mmol), Pd2(dba)3 (4.5 mg, 0.005 mmol), and NaOtBu (23.1
mg, 0.24 mmol) were added to the reaction vessel. The reaction vessel
was sealed and heated in a microwave at 90 °C for 20 min. The
reaction was allowed to cool to rt, and the solution was loaded onto a
1 g C18 SPE cartridge (preconditioned with MeCN [3 mL]). The
column was flushed with MeCN (3 mL), and the filtrate was
evaporated under a stream of nitrogen. The sample was dissolved in
DMSO (1 mL) and purified by MDAP (method B). The appropriate
fractions were evaporated, and the sample was dissolved in DMSO (1
mL) and purified by MDAP (method A) to give the title compound
1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (22h). 11 mg, 0.03
1
mmol, 6% yield. H NMR (600 MHz, DMSO-d6): δ 8.10 (s, 1 H),
7.72 (s, 1 H), 7.48 (d, J = 8.5 Hz, 1 H), 7.47 (s, 1 H), 7.03 (d, J = 8.5
Hz, 1 H), 4.23 (d, J = 7.5 Hz, 2 H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H),
3.55 (s, 3 H), 3.11 (t, J = 11.5 Hz, 2 H), 2.46 (s, 3 H), 2.10 (s, 3 H),
1.95 (ttt, J = 11.5, 7.5, 3.5, Hz, 1 H), 1.21 (dd, J = 11.5 Hz, 2 H), 1.14
(qd, J = 11.5, 3.5 Hz, 2 H). LC−MS (method F) m/z: 352 [(M +
H)+]; Rt: 0.55 min; 100% purity.
5-(5-Fluoro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]-
imidazole-2-yl)-1,3-dimethylpyridin-2(1H)-one (22i). 15 mg, 0.04
1
mmol, 9% yield. H NMR (600 MHz, DMSO-d6): δ 8.14 (s, 1 H),
7.74 (s, 1 H), 7.72 (dd, J = 8.5, 4.5 Hz, 1 H), 7.41 (dd, J = 9.5, 2.0 Hz,
1 H), 7.13 (ddd, J = 9.5, 8.5, 2.0 Hz, 1 H), 4.29 (d, J = 7.5 Hz, 2 H),
3.71 (dd, J = 11.5, 3.5 Hz, 2 H), 3.56 (s, 3 H), 3.11 (t, J = 11.5 Hz, 2
H), 2.10 (s, 3 H), 1.95 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.20 (dd, J =
11.5, Hz, 2 H), 1.15 (qd, J = 11.5, 3.5 Hz, 2 H). LC−MS (method F)
m/z: 356 [(M + H)+]; Rt: 0.65 min; 99% purity.
5-(5-Chloro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22j). 41 mg, 0.11
1
(8.9 mg, 0.02 mmol, 19% yield). H NMR (400 MHz, DMSO-d6): δ
1
mmol, 23% yield. H NMR (600 MHz, DMSO-d6): δ 8.15 (s, 1 H),
8.05 (d, J = 2.5 Hz, 1 H), 7.72−7.68 (m, 1 H), 7.41 (d, J = 9.0 Hz, 1
H), 6.83 (d, J = 2.5 Hz, 1 H), 6.76 (dd, J = 9.0, 2.5 Hz, 1 H), 4.20 (d,
J = 7.5 Hz, 2 H), 3.72 (dd, J = 11.5, 4.0 Hz, 2 H), 3.55 (s, 3 H), 3.11
(td, J = 11.5, 2.5 Hz, 2 H), 2.95 (s, 6 H), 2.10 (s, 3 H), 1.94 (ddd, J =
11.5, 7.5, 3.5 Hz, 1 H), 1.28−1.10 (m, 4 H); LC−MS (method F) m/
z: 381 [(M + H)+]; Rt: 0.50 min; 99% purity.
7.77−7.71 (m, 2 H), 7.67 (s, 1 H), 7.29 (d, J = 8.5 Hz, 1 H), 4.29 (d,
J = 7.5 Hz, 2 H), 3.71 (d, J = 11.5 Hz, 2 H), 3.56 (s, 3 H), 3.10 (t, J =
11.5 Hz, 2 H), 2.10 (s, 3 H), 1.98−1.89 (m, 1 H), 1.22−1.09 (m, 4
H). LC−MS (method F) m/z: 372, 374 [(M + H)+]; Rt: 0.79 min;
99% purity.
5-(6-Chloro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]-
General SNAr-Cyclization Method. To a solution of the
appropriately substituted 1-fluoro-2-nitrobenzene (83 mg, 0.52
mmol) in 2-MeTHF (0.5 mL) were added (tetrahydro-2H-pyran-4-
yl)methanamine (50 mg, 0.43 mmol) and DIPEA (0.23 mL, 1.30
mmol). The reaction vessel was sealed and heated in a microwave at
110 °C for 20 min. The solvent was removed under a stream of
nitrogen. To the dried material were added 1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carbaldehyde (72 mg, 0.48 mmol) and Na2S2O4
(227 mg, 1.30 mmol) as a solution in EtOH (1.0 mL), followed by
water (0.5 mL). The sealed vessels were heated at 100 °C for 18 h.
The solvent was removed under a stream of nitrogen. The reaction
mixture was partitioned between saturated aqueous NaHCO3 (3 mL)
and DCM (2 × 3 mL). The DCM extracts were evaporated under a
stream of nitrogen. The residue was dissolved in DMSO (1 mL) and
purified by MDAP (method B). The solvent was removed under a
stream of nitrogen to give the compounds 22e−22p.
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22k). 19 mg, 0.05
1
mmol, 11% yield. H NMR (600 MHz, DMSO-d6): δ 8.15 (s, 1 H),
7.89 (s, 1 H), 7.74 (s, 1 H), 7.62 (d, J = 8.5 Hz, 1 H), 7.23 (d, J = 8.5
Hz, 1 H), 4.29 (d, J = 7.5 Hz, 2 H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H),
3.55 (s, 3 H), 3.11 (t, J = 11.5 Hz, 2 H), 2.10 (s, 3 H), 1.93 (ttt, J =
11.5, 7.5, 3.5 Hz, 1 H), 1.20 (dd, J = 11.5 Hz, 2 H), 1.14 (qd, J = 11.5,
3.5 Hz, 2 H). LC−MS (method F) m/z: 372, 374 [(M + H)+]; Rt:
0.78 min; 99% purity.
2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahydro-
2H-pyran-4-yl)methyl)-1H-benzo[d]imidazole-5-carboxamide
(22l). 30 mg, 0.08 mmol, 16% yield. 1H NMR (600 MHz, DMSO-d6):
δ 8.18 (s, 1 H), 8.16 (s, 1 H), 7.98 (br s, 1 H), 7.83 (d, J = 8.5 Hz, 1
H), 7.75 (s, 1 H), 7.73 (d, J = 8.5 Hz, 1 H), 7.26 (br s, 1 H), 4.30 (d, J
= 7.5 Hz, 2 H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H), 3.56 (s, 3 H), 3.11 (t,
J = 11.5 Hz, 2 H), 2.11 (s, 3 H), 1.95 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H),
1.23−1.12 (m, 4 H). LC−MS (method F) m/z: 381 [(M + H)+]; Rt:
0.49 min; 100% purity.
5-(5-Methoxy-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo-
[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22e). 7 mg, 0.02
2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahydro-
1
mmol, 4% yield. H NMR (600 MHz, DMSO-d6): δ 8.09 (s, 1 H),
2H-pyran-4-yl)methyl)-1H-benzo[d]imidazole-6-carboxamide
1
7.72 (s, 1 H), 7.56 (d, J = 8.5 Hz, 1 H), 7.13 (d, J = 2.0 Hz, 1 H), 6.88
(dd, J = 8.5, 2.0 Hz, 1 H), 4.23 (d, J = 7.5 Hz, 2 H), 3.79 (s, 3 H),
3.71 (d, J = 11.5 Hz, 2 H), 3.55 (s, 3 H), 3.10 (t, J = 11.5 Hz, 2 H),
2.10 (s, 3 H), 1.94 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.20 (d, J = 11.5
Hz, 2 H), 1.13 (qd, J = 11.5, 3.5 Hz, 2 H). LC−MS (method F) m/z:
368 [(M + H)+]; Rt: 0.51 min; 96% purity.
(22m). 1 mg, 0.003 mmol, 1% yield. H NMR (600 MHz, DMSO-
d6): δ 8.22 (s, 1 H), 8.18 (s, 1 H), 8.00 (br s, 1 H), 7.79 (d, J = 8.5
Hz, 1 H), 7.77 (s, 1 H), 7.63 (d, J = 8.5 Hz, 1 H), 7.35 (br s, 1 H),
4.32 (d, J = 7.5 Hz, 2 H), 3.72 (dd, J = 11.5, 3.5 Hz, 2 H), 3.56 (s, 3
H), 3.12 (t, J = 11.5 Hz, 2 H), 2.11 (s, 3 H), 2.00 (ttt, J = 11.5, 7.5,
3.5 Hz, 1 H), 1.24−1.13 (m, 4 H). LC−MS (method F) m/z: 381
[(M + H)+]; Rt: 0.47 min; 100% purity.
5-(6-Methoxy-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo-
[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (22f). 8 mg, 0.02
2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahydro-
1
mmol, 4% yield. H NMR (600 MHz, DMSO-d6): δ 8.08 (s, 1 H),
2H-pyran-4-yl)methyl)-1H-benzo[d]imidazole-5-carbonitrile (22n).
1
7.71 (s, 1 H), 7.48 (d, J = 8.5 Hz, 1 H), 7.22 (s, 1 H), 6.83 (dd, J =
8.5, 2.0 Hz, 1 H), 4.24 (d, J = 7.5 Hz, 2 H), 3.83 (s, 3 H), 3.71 (d, J =
11.5 Hz, 2 H), 3.55 (s, 3 H), 3.11 (t, J = 11.5 Hz, 2 H), 2.10 (s, 3 H),
1.93 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.20 (d, J = 11.5 Hz, 2 H), 1.19−
54 mg, 0.15 mmol, 31% yield. H NMR (600 MHz, DMSO-d6): δ
8.21 (s, 1 H), 8.16 (s, 1 H), 7.94 (d, J = 8.5 Hz, 1 H), 7.77 (s, 1 H),
7.67 (d, J = 8.5 Hz, 1 H), 4.36 (d, J = 7.5 Hz, 2 H), 3.71 (dd, J = 11.5,
3.5 Hz, 2 H), 3.56 (s, 3 H), 3.10 (td, J = 11.5, 3.5 Hz, 2 H), 2.11 (s, 3
W
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
Article
H), 1.93 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.22−1.12 (m, 4 H). LC−
MS (method F) m/z: 363 [(M + H)+]; Rt: 0.74 min; 97% purity.
2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahydro-
2H-pyran-4-yl)methyl)-1H-benzo[d]imidazole-6-carbonitrile (22o).
JohnPhos (9 mg, 0.03 mmol), and NaOtBu (23 mg, 0.24 mmol) were
suspended in toluene (2 mL) within a sealed microwave vial. The
reaction was heated in a microwave at 90 °C for 10 min. The reaction
mixture was filtered over a Celite pad and washed with EtOAc (20
mL). The solvent was removed in vacuo, and the residue was
dissolved in 1:1 DMSO/MeOH (0.6 mL) and purified by MDAP
(method B). The solvent was evaporated in vacuo to give the title
1
36 mg, 0.10 mmol, 20% yield. H NMR (600 MHz, DMSO-d6): δ
8.40 (s, 1 H), 8.24−8.22 (m, 1 H), 7.79 (s, 1 H), 7.77 (d, J = 8.5 Hz,
1 H), 7.60 (d, J = 8.5 Hz, 1 H), 4.37 (d, J = 7.5 Hz, 2 H), 3.72 (dd, J
= 11.5, 3.5 Hz, 2 H), 3.56 (s, 3 H), 3.12 (td, J = 11.5, 3.5 Hz, 2 H),
2.11 (s, 3 H), 1.97 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.22−1.13 (m, 4
H). LC−MS (method F) m/z: 363 [(M + H)+]; Rt: 0.74 min; 95%
purity.
1
compound (29 mg, 0.07 mmol, 30% yield). H NMR (400 MHz,
CDCl3): δ 7.72 (d, J = 2.5 Hz, 1 H), 7.52−7.49 (m, 1 H), 7.23 (d, J =
8.5 Hz, 1 H), 6.90 (d, J = 2.0 Hz, 1 H), 6.70 (dd, J = 8.5, 2.0 Hz, 1
H), 4.08 (d, J = 7.5 Hz, 2 H), 3.89 (dd, J = 11.5, 3.5 Hz, 2 H), 3.65 (s,
3 H), 3.38−3.32 (m, 4 H), 3.24 (td, J = 11.5, 2.0 Hz, 2 H), 2.25 (s, 3
H), 2.16−2.03 (m, 5 H), 1.40−1.20 (m, 4 H); LC−MS (method A)
m/z: 407 [(M + H)+]; Rt: 0.98 min; 97% purity.
1,3-Dimethyl-5-(5-(methylsulfonyl)-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (22p). 11
1
mg, 0.03 mmol, 6% yield. H NMR (600 MHz, DMSO-d6): δ 8.20
(d, J = 2.5 Hz, 1 H), 8.16−8.15 (m, 1 H), 7.99 (d, J = 8.5 Hz, 1 H),
7.81 (d, J = 8.5 Hz, 1 H), 7.77 (s, 1 H), 4.37 (d, J = 7.5 Hz, 2 H), 3.72
(dd, J = 11.5, 3.5 Hz, 2 H), 3.57 (s, 3 H), 3.24 (s, 3 H), 3.11 (td, J =
11.5, 3.5 Hz, 2 H), 2.11 (s, 3 H), 1.95 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H),
1.24−1.13 (m, 4 H). LC−MS (method F) m/z: 416 [(M + H)+]; Rt:
0.64 min; 99% purity.
1,3-Dimethyl-5-(6-(pyrrolidin-1-yl)-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (23d). 5-
(6-Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]-
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one 22b (100 mg, 0.24
mmol), pyrrolidine (0.02 mL, 0.24 mmol), Pd2(dba)3 (8.8 mg, 0.01
mmol), JohnPhos (8.6 mg, 0.03 mmol), and NaOtBu (23.1 mg, 0.24
mmol) were dissolved in toluene (2 mL). The reaction was heated in
a microwave at 90 °C for 10 min. The reaction was heated in a
microwave at 90 °C for a further 20 min. The cooled reaction mixture
was filtered through Celite washing with EtOAc. The filtrate was
concentrated, and the crude material was purified by loading onto a
12 g silica column and eluting using a graduating solvent system of 0−
30% EtOH in EtOAc. The appropriate fractions were combined,
concentrated, and purified by MDAP (method A). Concentration of
the collected fractions gave the title compound as a tan solid (22 mg,
0.05 mmol, 23% yield). 1H NMR (400 MHz, CDCl3): δ 8.00 (s, 1 H),
7.63 (d, J = 8.5 Hz, 1 H), 7.43 (s, 1 H), 6.73 (d, J = 8.5 Hz, 1 H), 6.34
(s, 1 H), 4.09 (d, J = 7.5 Hz, 2 H), 3.92 (dd, J = 11.5, 3.5 Hz, 2 H),
3.67 (s, 3 H), 3.41−3.34 (m, 4 H), 3.26 (t, J = 11.5 Hz, 2 H), 2.25 (s,
3 H), 2.18−2.05 (m, 5 H), 1.42−1.22 (m, 4 H); LC−MS (method F)
m/z: 407 [(M + H)+]; Rt: 0.64 min; 100% purity.
5-(5-(Cyclobutylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-
1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (23a). A
suspension of 5-(5-bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one 22a (50 mg,
0.12 mmol), NaOtBu (34.6 mg, 0.36 mmol), BrettPhos (6.5 mg, 0.01
mmol), and BrettPhos Pd G1 precatalyst (9.5 mg, 0.01 mmol) in 1,4-
dioxane (1 mL) was prepared in a vial containing cyclobutylamine
hydrochloride (15 mg, 0.14 mmol). A stirrer bar was added, and the
vial was sealed, sonicated, and heated in a microwave at 110 °C for 30
min. The reaction was allowed to cool and loaded onto a 1 g C18 SPE
cartridge (preconditioned with MeCN). The cartridge was flushed
with MeCN (2 mL) and MeOH (2 mL), and the solvent was
removed under a stream of nitrogen. The sample was dissolved in
DMSO (1 mL) and purified by MDAP (method B). The solvent was
removed using a plate dryer to give the title compound (5 mg, 0.01
1
mmol, 9% yield). H NMR (600 MHz, DMSO-d6): δ 8.04 (s, 1 H),
1,3-Dimethyl-5-(5-morpholino-1-((tetrahydro-2H-pyran-4-yl)-
methyl)-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (23e). 5-(5-
Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one 22a (100 mg, 0.24 mmol),
morpholine (0.03 mL, 0.34 mmol), Pd2(dba)3 (9 mg, 0.01 mmol),
JohnPhos (9 mg, 0.03 mmol), and NaOtBu (23 mg, 0.24 mmol) were
suspended in toluene (2 mL) within a sealed microwave vial. The
reaction mixture was heated in a microwave at 90 °C for 10 min. The
reaction mixture was cooled to rt, filtered over a Celite pad, and
washed with EtOAc (20 mL). The solvent was removed in vacuo, and
the residue was dissolved in 1:1 DMSO/MeOH (0.6 mL) and
purified by MDAP (method B). The solvent was evaporated in vacuo
to give the title compound (56 mg, 0.13 mmol, 55% yield). 1H NMR
(400 MHz, CDCl3): δ 7.73 (d, J = 2.5 Hz, 1 H), 7.51−7.48 (m, 1 H),
7.29 (d, J = 9.0 Hz, 1 H), 7.27 (d, J = 2.5 Hz, 1 H), 7.04 (dd, J = 9.0,
2.5 Hz, 1 H), 4.10 (d, J = 7.5 Hz, 2 H), 3.94−3.87 (m, 6 H), 3.66 (s, 3
H), 3.25 (td, J = 11.5, 2.0 Hz, 2 H), 3.20−3.17 (m, 4 H), 2.25 (s, 3
H), 2.09 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.39−1.22 (m, 4 H); LC−
MS (method F) m/z: 423 [(M + H)+]; Rt: 0.55 min; 100% purity.
1,3-Dimethyl-5-(6-morpholino-1-((tetrahydro-2H-pyran-4-yl)-
methyl)-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (23f). 5-(6-
Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one 22b (100 mg, 0.24 mmol),
morpholine (0.03 mL, 0.34 mmol), Pd2(dba)3 (9 mg, 0.01 mmol),
JohnPhos (9 mg, 0.03 mmol), and NaOtBu (23 mg, 0.24 mmol) were
suspended in toluene (2 mL) within a sealed microwave vial. The
reaction mixture was heated in a microwave at 90 °C for 10 min. The
reaction mixture was allowed to cool to rt, filtered over Celite, and
washed with EtOAc (50 mL). The solvent was removed in vacuo, and
the sample was dissolved in 1:1 DMSO/MeOH (0.6 mL) and purified
by MDAP (method B). The solvent was evaporated in vacuo to give
7.69 (s, 1 H), 7.34 (d, J = 8.5 Hz, 1 H), 6.61−6.57 (m, 2 H), 5.57 (d,
J = 7.0 Hz, 1 H), 4.14 (d, J = 7.5 Hz, 2 H), 3.88−3.79 (m, 1 H), 3.71
(d, J = 11.5 Hz, 2 H), 3.54 (s, 3 H), 3.10 (t, J = 11.5 Hz, 2 H), 2.38−
2.31 (m, 2 H), 2.09 (s, 3 H), 1.97−1.88 (m, 1 H), 1.86−1.77 (m, 2
H), 1.77−1.69 (m, 2 H), 1.20 (d, J = 11.5 Hz, 2 H), 1.11 (q, J = 11.5
Hz, 2 H); LC−MS (method F) m/z: 407 [(M + H)+]; Rt: 0.61 min;
95% purity.
5-(6-(Cyclobutylamino)-1-((tetrahydro-2H-pyran-4-yl)methyl)-
1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (23b). A
suspension of 5-(6-bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one 22b (50 mg,
0.12 mmol), NaOtBu (23.1 mg, 0.24 mmol), BrettPhos (6.5 mg, 0.01
mmol), and BrettPhos Pd G1 precatalyst (9.6 mg, 0.012 mmol), in
1,4-dioxane (1 mL), was prepared in a vial containing cyclobutyl-
amine hydrochloride (12.9 mg, 0.12 mmol). A stirrer bar was added,
and the vial was sealed and heated in a microwave at 120 °C for 30
min. The reaction was allowed to cool and loaded onto a 1 g C18 SPE
cartridge (preconditioned with MeCN). The cartridge was flushed
with MeCN (2 mL) and MeOH (2 mL), and the solvent was
removed under a stream of nitrogen. The sample was dissolved in
DMSO (1 mL) and purified by MDAP (method B). The solvent was
removed using a plate dryer to give the title compound (11 mg, 0.03
mmol, 20% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.00 (d, J = 2.5
Hz, 1 H), 7.69−7.66 (m, 1 H), 7.29 (d, J = 8.5 Hz, 1 H), 6.54−6.50
(m, 2 H), 5.77 (d, J = 7.0 Hz, 1 H), 4.12 (d, J = 7.5 Hz, 2 H), 3.98−
3.87 (m, 1 H), 3.72 (d, J = 11.5 Hz, 2 H), 3.57−3.52 (m, 3 H), 3.12
(td, J = 11.5, 3.0 Hz, 2 H), 2.44−2.34 (m, 2 H), 2.09 (s, 3 H), 1.93
(d, J = 11.0 Hz, 1 H), 1.87−1.69 (m, 4 H), 1.28−1.09 (m, 4 H); LC−
MS (method F) m/z: 407 [(M + H)+]; Rt: 0.61 min; 97% purity.
1,3-Dimethyl-5-(5-(pyrrolidin-1-yl)-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one (23c). 5-(5-
Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one 22a (100 mg, 0.240 mmol),
pyrrolidine (0.02 mL, 0.24 mmol), Pd2(dba)3 (9 mg, 0.01 mmol),
1
the title compound (29 mg, 0.07 mmol, 29% yield). H NMR (400
MHz, CDCl3): δ 7.70 (d, J = 2.5 Hz, 1 H), 7.65 (d, J = 8.5 Hz, 1 H),
7.49−7.46 (m, 1 H), 7.02 (dd, J = 8.5, 2.0 Hz, 1 H), 6.80 (d, J = 2.0
Hz, 1 H), 4.09 (d, J = 7.5 Hz, 2 H), 3.96−3.88 (m, 6 H), 3.65 (s, 3
X
DOI: 10.1021/acs.jmedchem.9b01670
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
H), 3.30−3.20 (m, 6 H), 2.25 (s, 3 H), 2.07 (ttt, J = 11.5, 7.5, 3.5 Hz,
1 H), 1.40−1.23 (m, 4 H); LC−MS (method A) m/z: 423 [(M +
H)+]; Rt: 0.76 min; 100% purity.
22% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.05 (s, 1 H), 7.70 (s,
1 H), 7.42 (d, J = 8.5 Hz, 1 H), 6.80 (d, J = 2.0 Hz, 1 H), 6.76 (dd, J
= 8.5, 2.0 Hz, 1 H), 4.16 (d, J = 7.5 Hz, 2 H), 3.72 (dd, J = 11.5, 3.5
Hz, 2 H), 3.55 (s, 3 H), 3.54−3.51 (m, 2 H), 3.45 (t, J = 6.0 Hz, 2 H),
3.11 (t, J = 11.5 Hz, 2 H), 2.66−2.62 (m, 2 H), 2.48−2.45 (m, 2 H),
2.26 (s, 3 H), 2.09 (s, 3 H), 1.99−1.89 (m, 3 H), 1.22 (dd, J = 11.5,
3.5 Hz, 2 H), 1.13 (qd, J = 11.5, 3.5 Hz, 2 H); LC−MS (method F)
m/z: 450 [(M + H)+]; Rt: 0.35 min; 95% purity.
1,3-Dimethyl-5-(6-(4-methyl-1,4-diazepan-1-yl)-1-((tetrahydro-
2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-
one (23j). 1-Methyl-1,4-diazepane (14 mg, 0.120 mmol) was
dissolved in 1,4-dioxane (1.5 mL) and added to 5-(6-bromo-1-
((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-
dimethylpyridin-2(1H)-one 22b (50 mg, 0.12 mmol), JohnPhos (4.6
mg, 0.02 mmol), NaOtBu (23.1 mg, 0.24 mmol), and Pd2(dba)3 (4.5
mg, 0.005 mmol). The reaction vessel was sealed and heated in a
microwave at 110 °C for 20 min. After cooling the reaction to rt, the
reaction mixture was loaded onto a 1 g C18 SPE cartridge
(preconditioned with MeCN [3 mL]). The cartridge was flushed
with MeCN (3 mL), and the solvent was removed under a stream of
nitrogen. The sample was dissolved in DMSO (1 mL) and purified by
MDAP (method B). The solvent was evaporated under a stream of
nitrogen to give the title compound (26 mg, 0.06 mmol, 43% yield).
1H NMR (600 MHz, DMSO-d6): δ 8.04 (s, 1 H), 7.69 (s, 1 H), 7.39
(d, J = 8.5 Hz, 1 H), 6.78 (d, J = 2.0 Hz, 1 H), 6.72 (dd, J = 8.5, 2.0
Hz, 1 H), 4.19 (d, J = 7.5 Hz, 2 H), 3.72 (dd, J = 11.5, 3.5 Hz, 2 H),
3.66−3.62 (m, 2 H), 3.54 (s, 3 H), 3.51 (t, J = 6.0 Hz, 2 H), 3.10 (t, J
= 11.5 Hz, 2 H), 2.89−2.81 (m, 2 H), 2.71−2.65 (m, 2 H), 2.42 (s, 3
H), 2.09 (s, 3 H), 2.05−1.98 (m, 2 H), 1.91 (ttt, J = 11.5, 7.5, 3.5 Hz,
1 H), 1.21 (dd, J = 11.5, 3.5 Hz, 2 H), 1.16 (qd, J = 11.5, 3.5 Hz, 2
H); LC−MS (method F) m/z: 450 [(M + H)+]; Rt: 0.35 min; 98%
purity.
1-(2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-5-yl)-N-methylpi-
peridine-4-carboxamide (23k). A stock solution of 5-(5-bromo-1-
((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-
dimethylpyridin-2(1H)-one 22a (300 mg, 0.12 mmol) was prepared
in 1,4-dioxane (3 mL), and 0.5 mL aliquoted into a vial containing N-
methylpiperidine-4-carboxamide (20 mg, 0.14 mmol). To the vial was
added 1,4-dioxane (0.5 mL) along with NaOtBu (34.6 mg, 0.36
mmol) and RuPhos Pd G2 precatalyst (18.7 mg, 0.024 mmol). A
stirrer bar was added, and the vial was sonicated and capped before
heating in a microwave at 110 °C for 30 min. The reaction mixture
was allowed to cool and then loaded onto a 1 g C18 SPE cartridge
(preconditioned with MeCN). The cartridge was flushed with MeCN
(2 mL) and MeOH (2 mL), and the solvent was removed under a
stream of nitrogen. The sample was dissolved in DMSO (1 mL) and
purified by MDAP (method B). The solvent was removed using a
plate dryer to give the title compound (15 mg, 0.03 mmol, 24% yield).
1H NMR (600 MHz, DMSO-d6): δ 8.07 (d, J = 2.5 Hz, 1 H), 7.74 (q,
2-((2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-5-yl)amino)-N,N-
dimethylacetamide (23g). 5-(5-Bromo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-
one 22a (50 mg, 0.12 mmol), NaOtBu (23.1 mg, 0.24 mmol),
BrettPhos (8.3 mg, 0.02 mmol), and BrettPhos Pd G1 precatalyst (4.8
mg, 0.006 mmol) were added to 2-amino-N,N-dimethylacetamide
acetate (19 mg, 0.12 mmol) and suspended in 1,4-dioxane (1.5 mL).
DIPEA (0.02 mL) was added to the mixture, and the reaction vessel
was sealed and heated in a microwave at 110 °C for 30 min. After
cooling the reaction to rt, the reaction mixture was loaded onto a 1 g
C18 SPE cartridge (preconditioned with MeCN [3 mL]). The
cartridge was flushed with MeCN (3 mL), and the solvent was
removed under a stream of nitrogen. The sample was dissolved in
DMSO (1 mL) and purified by MDAP (method B). The solvent was
dried under a stream of nitrogen to give the title compound (25 mg,
0.06 mmol, 43% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.05 (s, 1
H), 7.69 (s, 1 H), 7.37 (d, J = 8.5 Hz, 1 H), 6.77−6.73 (m, 2 H), 5.30
(t, J = 5.0 Hz, 1 H), 4.16 (d, J = 7.5 Hz, 2 H), 3.89 (d, J = 5.0 Hz, 2
H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H), 3.54 (s, 3 H), 3.11 (t, J = 11.5
Hz, 2 H), 3.05 (s, 3 H), 2.89 (s, 3 H), 2.09 (s, 3 H), 1.93 (ttt, J =
11.5, 7.5, 3.5 Hz, 1 H), 1.20 (dd, J = 11.5, 3.5 Hz, 2 H), 1.12 (qd, J =
11.5, 3.5 Hz, 2 H); LC−MS (method F) m/z: 438 [(M + H)+]; Rt:
0.48 min; 95% purity.
2-((2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-6-yl)amino)-N,N-
dimethylacetamide (23h). 5-(6-Bromo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-
one 22b (50 mg, 0.12 mmol), BrettPhos (8.3 mg, 0.02 mmol),
BrettPhos Pd G1 precatalyst (4.8 mg, 0.006 mmol), and NaOtBu
(23.1 mg, 0.24 mmol) were added to 2-amino-N,N-dimethylaceta-
mide acetate (19 mg, 0.12 mmol) suspended in 1,4-dioxane (1.5 mL).
The reaction vessel was sealed and heated in a microwave at 110 °C
for 30 min. After cooling the reaction to rt, BrettPhos (8.3 mg, 0.02
mmol) and BrettPhos Pd G1 precatalyst (4.8 mg, 0.006 mmol) were
added. The reaction vessel was sealed and heated in a microwave at
110 °C for 30 min. After cooling the reaction to rt, the reaction
mixture was loaded onto a 1 g C18 SPE cartridge (preconditioned
with MeCN [3 mL]). The cartridge was flushed with MeCN (3 mL),
and the solvent was removed under a stream of nitrogen. The sample
was dissolved in DMSO (1 mL) and purified by MDAP (method B).
The solvent was evaporated under a stream of nitrogen to give the
title compound (12 mg, 0.03 mmol, 21% yield). 1H NMR (600 MHz,
DMSO-d6): δ 8.03 (s, 1 H), 7.69 (s, 1 H), 7.32 (d, J = 8.5 Hz, 1 H),
6.74 (s, 1 H), 6.68 (d, J = 8.5 Hz, 1 H), 5.47 (t, J = 5.0 Hz, 1 H), 4.14
(d, J = 7.5 Hz, 2 H), 3.94 (d, J = 5.0 Hz, 2 H), 3.73 (dd, J = 11.5, 3.5
Hz, 2 H), 3.54 (s, 3 H), 3.12 (t, J = 11.5 Hz, 2 H), 3.07 (s, 3 H), 2.89
(s, 3 H), 2.09 (s, 3 H), 1.94 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.23 (dd,
J = 11.5, 3.5 Hz, 2 H), 1.16 (qd, J = 11.5, 3.5 Hz, 2 H); LC−MS
(method F) m/z: 438 [(M + H)+]; Rt: 0.48 min; 100% purity.
1,3-Dimethyl-5-(5-(4-methyl-1,4-diazepan-1-yl)-1-((tetrahydro-
2H-pyran-4-yl)methyl)-1H-benzo[d]imidazole-2-yl)pyridin-2(1H)-
one (23i). 5-(5-Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazole-2-yl)-1,3-dimethylpyridin-2(1H)-one 22a (50
mg, 0.12 mmol), NaOtBu (23.1 mg, 0.24 mmol), and RuPhos Pd
G2 precatalyst (2.4 mg, 0.003 mmol) were added to 1-methyl-1,4-
diazepane (14 mg, 0.12 mmol) in 1,4-dioxane. The reaction vessel was
sealed and heated in a microwave at 110 °C for 30 min. After cooling
the reaction to rt, the RuPhos Pd G2 precatalyst (2.4 mg, 0.003
mmol) was added to the reaction mixture. The reaction vessel was
sealed and heated in a microwave at 110 °C for 30 min. After cooling
the reaction to rt, the reaction mixture was loaded onto a 1 g C18 SPE
cartridge (preconditioned with MeCN [3 mL]). The cartridge was
flushed with MeCN (3 mL), and the solvent was removed under a
stream of nitrogen. The sample was dissolved in DMSO (1 mL) and
purified by MDAP (method B). The solvent was evaporated under a
stream of nitrogen to give the title compound (13 mg, 0.03 mmol,
J = 4.5 Hz, 1 H), 7.71−7.69 (m, 1 H), 7.50 (d, J = 8.5 Hz, 1 H), 7.07
(d, J = 2.0 Hz, 1 H), 7.01 (dd, J = 8.5, 2.0 Hz, 1 H), 4.20 (d, J = 7.5
Hz, 2 H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H), 3.61 (d, J = 12.0 Hz, 2 H),
3.55 (s, 3 H), 3.11 (t, J = 11.5 Hz, 2 H), 2.63 (td, J = 12.0, 3.5 Hz, 2
H), 2.58 (d, J = 4.5 Hz, 3 H), 2.21 (tt, J = 11.5, 4.0 Hz, 1 H), 2.10 (s,
3 H), 1.93 (ttt, J = 11.5, 7.5, 3.5 Hz, 1 H), 1.81−1.68 (m, 4 H), 1.21
(dd, J = 11.5, 3.5 Hz, 2 H), 1.13 (qd, J = 11.5, 3.5 Hz, 2 H); LC−MS
(method F) m/z: 478 [(M + H)+]; Rt: 0.47 min; 100% purity.
1-(2-(1,5-Dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-6-yl)-N-methylpi-
peridine-4-carboxamide (23l). To a vial containing N-methylpiper-
idine-4-carboxamide (20 mg, 0.14 mmol) was added 1.0 mL of a stock
solution of 5-(6-bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one 22b (150
mg, 0.12 mmol) in 1,4-dioxane (3 mL). Pd2(dba)3 (11 mg, 0.01
mmol), NaOtBu (34.6 mg, 0.36 mmol), JohnPhos (7.2 mg, 0.02
mmol), and a stirrer bar were added to the vial, which was sealed,
sonicated, and heated in a microwave at 90 °C for 20 min. The
reaction mixture was allowed to cool and then loaded onto a 1 g C18
SPE cartridge (preconditioned with MeCN). The cartridge was
Y
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Journal of Medicinal Chemistry
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flushed with MeCN (2 mL) and MeOH (2 mL), and the solvent was
removed under a stream of nitrogen. The sample was dissolved in
DMSO (1 mL) and purified by MDAP (method B). The solvent was
removed using a plate dryer to give the title compound (18 mg, 0.04
mmol, 28% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.06 (d, J = 2.5
Hz, 1 H), 7.75−7.68 (m, 2 H), 7.42 (d, J = 8.5 Hz, 1 H), 7.10 (d, J =
2.0 Hz, 1 H), 6.93 (dd, J = 8.5, 2.0 Hz, 1 H), 4.22 (d, J = 7.5 Hz, 2
H), 3.75−3.68 (m, 4 H), 3.55 (s, 3 H), 3.11 (td, J = 11.5, 2.0 Hz, 2
H), 2.69 (td, J = 11.5, 3.5 Hz, 3 H), 2.59 (d, J = 4.5 Hz, 4 H), 2.24 (tt,
J = 11.5, 4.0 Hz, 1 H), 2.10 (s, 3 H), 1.98−1.85 (m, 1 H), 1.85−1.67
(m, 4 H), 1.26−1.10 (m, 5 H); LC−MS (method F) m/z: 478 [(M +
H)+]; Rt: 0.44 min; 100% purity.
(methylsulfonyl)pyrrolidine (21 mg, 0.14 mmol). To the vial was
added further 1,4-dioxane (0.4 mL) along with the RuPhos Pd G2
precatalyst (18.7 mg, 0.02 mmol) and NaOtBu (34.6 mg, 0.36 mmol).
A stirrer bar was added, and the vial was sonicated and capped before
heating in a microwave at 110 °C for 30 min. The reaction mixture
was allowed to cool and then loaded onto a 1 g C18 SPE cartridge
(preconditioned with MeCN). The cartridge was flushed with MeCN
(2 mL) and MeOH (2 mL), and the solvent was removed under a
stream of nitrogen. The sample was dissolved in DMSO (1 mL) and
purified by MDAP (method B). The solvent was removed using a
plate dryer to give the title compound (21 mg, 0.04 mmol, 32% yield).
1H NMR (600 MHz, DMSO-d6): δ 8.08 (s, 1 H), 7.71 (s, 1 H), 7.51
1,3-Dimethyl-5-(5-(4-(methylamino)piperidin-1-yl)-1-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-
2(1H)-one (23m). 5-(5-Bromo-1-((tetrahydro-2H-pyran-4-yl)-
methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one
22a (50 mg, 0.12 mmol), NaOtBu (23.1 mg, 0.240 mmol), and
RuPhos Pd G2 precatalyst (2.4 mg, 0.003 mmol) were added to tert-
butyl methyl(piperidin-4-yl)carbamate (26 mg, 0.12 mmol) in 1,4-
dioxane (1.5 mL). The reaction vessel was sealed and heated in a
microwave at 110 °C for 30 min. After cooling the reaction to rt, the
RuPhos Pd G2 precatalyst (2.4 mg, 0.003 mmol) was added. The
reaction vessel was sealed and heated in a microwave at 110 °C for 30
min. After cooling the reaction to rt, the reaction mixture was loaded
onto a 1 g C18 SPE cartridge (preconditioned with MeCN [3 mL]).
The cartridge was flushed with MeCN (3 mL), and the solvent was
removed under a stream of nitrogen. HCl (4 M) in dioxane (0.5 mL)
was added to the sample, and the reaction was left overnight. The
solvent was removed under a stream of nitrogen. The sample was
dissolved in DMSO (1 mL) and purified by MDAP (method B). The
solvent was dried under a stream of nitrogen to give the title
(d, J = 8.5 Hz, 1 H), 6.79 (s, 1 H), 6.73 (d, J = 8.5 Hz, 1 H), 4.20 (d, J
= 7.5 Hz, 2 H), 4.10 (quin, J = 7.0 Hz, 1 H), 3.71 (dd, J = 11.5, 3.5
Hz, 2 H), 3.65−3.60 (m, 2 H), 3.55 (s, 3 H), 3.47−3.40 (m, 2 H),
3.10 (t, J = 11.5 Hz, 2 H), 3.06 (s, 3 H), 2.42−2.37 (m, 1 H), 2.10 (s,
3 H), 1.98−1.89 (m, 1 H), 1.19 (dd, J = 11.5, 3.5 Hz, 2 H), 1.13 (qd,
J = 11.5, 3.5 Hz, 2 H); LC−MS (method F) m/z: 485 [(M + H)+];
Rt: 0.52 min; 100% purity.
1,3-Dimethyl-5-(6-(3-(methylsulfonyl)pyrrolidin-1-yl)-1-((tetra-
hydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-
2(1H)-one (23p). A stock solution of 5-(6-bromo-1-((tetrahydro-2H-
pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-
2(1H)-one 22b (500 mg, 0.12 mmol) was prepared in 1,4-dioxane (5
mL), and an aliquot (0.5 mL) was added to a vial containing 3-
(methylsulfonyl)pyrrolidine (18 mg, 0.12 mmol). To the vial was
added further 1,4-dioxane (0.5 mL) along with JohnPhos (7.2 mg,
0.02 mmol), NaOtBu (34.6 mg, 0.36 mmol), and Pd2(dba)3 (11.0 mg,
0.01 mmol). A stirrer bar was added, and the vial was sonicated and
capped before heating in a microwave at 90 °C for 20 min. The
reaction mixture was allowed to cool and then loaded onto a 1 g C18
SPE cartridge (preconditioned with MeCN). The cartridge was
flushed with MeCN (2 mL) and MeOH (2 mL), and the solvent was
removed under a stream of nitrogen. The sample was purified by
MDAP (method B). The solvent was removed using a plate dryer to
1
compound (12 mg, 0.03 mmol, 20% yield). H NMR (600 MHz,
DMSO-d6): δ 8.07 (s, 1 H), 7.70 (s, 1 H), 7.49 (d, J = 9.0 Hz, 1 H),
7.07 (d, J = 2.0 Hz, 1 H), 7.00 (dd, J = 9.0, 2.0 Hz, 1 H), 4.20 (d, J =
7.5 Hz, 2 H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H), 3.57−3.51 (m, 5 H),
3.11 (t, J = 11.5 Hz, 2 H), 2.70 (t, J = 12.0 Hz, 2 H), 2.47−2.40 (m, 1
H), 2.32 (s, 3 H), 2.09 (s, 3 H), 1.98−1.88 (m, 3 H), 1.44−1.36 (m, 2
H), 1.21 (d, J = 11.5 Hz, 2 H), 1.13 (qd, J = 11.5, 3.5 Hz, 2 H); LC−
MS (method A) m/z: 450 [(M + H)+]; Rt: 0.74 min; 96% purity.
1,3-Dimethyl-5-(6-(4-(methylamino)piperidin-1-yl)-1-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-
2(1H)-one (23n). tert-Butyl methyl(piperidin-4-yl)carbamate (26 mg,
0.12 mmol) was dissolved in 1,4-dioxane (1.5 mL) and added to 5-(6-
bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one 22b (50 mg, 0.12 mmol),
JohnPhos (4.6 mg, 0.02 mmol), NaOtBu (23.1 mg, 0.24 mmol),
and Pd2(dba)3 (4.5 mg, 0.005 mmol). The reaction vessel was sealed
and heated in a microwave at 110 °C for 30 min. After cooling the
reaction to rt, the reaction mixture was loaded onto a 1 g C18 SPE
cartridge (preconditioned with MeCN [3 mL]). The cartridge was
flushed with MeCN (3 mL), and the solvent was removed under a
stream of nitrogen. HCl (4 M) in dioxane (0.5 mL) was added to the
sample, and the reaction was left for 1 h. The solvent was removed
under a stream of nitrogen. The sample was dissolved in DMSO (1
mL) and purified by MDAP (method B). The solvent was dried under
a stream of nitrogen to give the title compound (13 mg, 0.03 mmol,
21% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.05 (s, 1 H), 7.70 (s,
1 H), 7.41 (d, J = 9.0 Hz, 1 H), 7.08 (s, 1 H), 6.92 (d, J = 9.0 Hz, 1
H), 4.21 (d, J = 7.5 Hz, 2 H), 3.71 (dd, J = 11.5, 3.5 Hz, 2 H), 3.61
(d, J = 12.0 Hz, 2 H), 3.54 (s, 3 H), 3.11 (t, J = 11.5 Hz, 2 H), 2.76 (t,
J = 12.0 Hz, 2 H), 2.44−2.38 (m, 1 H), 2.30 (s, 3 H), 2.09 (s, 3 H),
1.95−1.88 (m, 3 H), 1.43−1.34 (m, 2 H), 1.21 (d, J = 11.5 Hz, 2 H),
1.15 (qd, J = 11.5, 3.5 Hz, 2 H); LC−MS (method F) m/z: 450 [(M
+ H)+]; Rt: 0.35 min; 98% purity.
1
give the title compound (9 mg, 0.02 mmol, 14% yield). H NMR
(600 MHz, DMSO-d6): δ 8.06 (s, 1 H), 7.71 (s, 1 H), 7.44 (d, J = 8.5
Hz, 1 H), 6.77 (s, 1 H), 6.64 (d, J = 8.5 Hz, 1 H), 4.22 (d, J = 7.5 Hz,
2 H), 4.14 (quin, J = 7.0 Hz, 1 H), 3.72 (d, J = 11.5 Hz, 2 H), 3.68−
3.62 (m, 2 H), 3.55 (s, 3 H), 3.53−3.47 (m, 2 H), 3.11 (t, J = 11.5
Hz, 2 H), 3.08 (s, 3 H), 2.44−2.37 (m, 2 H), 2.09 (s, 3 H), 1.98−1.88
(m, 1 H), 1.27−1.10 (m, 4 H); LC−MS (method F) m/z: 485 [(M +
H)+]; Rt: 0.49 min; 99% purity.
5-(5-(1,1-Dioxidothiomorpholino)-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-
one (23q). 5-(5-Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one 22a (100
mg, 0.24 mmol), thiomorpholine 1,1-dioxide (39 mg, 0.29 mmol),
Pd2(dba)3 (9 mg, 0.01 mmol), JohnPhos (9 mg, 0.03 mmol), and
NaOtBu (23 mg, 0.24 mmol) were suspended in toluene (2 mL)
within a sealed microwave vial. The reaction mixture was heated in a
microwave at 90 °C for 10 min. The reaction mixture was cooled to rt,
filtered over Celite, and washed with EtOAc (50 mL). The solvent
was removed in vacuo, and the sample was dissolved in 1:1 DMSO/
MeOH (0.6 mL) and purified by MDAP (method B). The solvent
was evaporated in vacuo to give the title compound (5 mg, 0.01
mmol, 4% yield). 1H NMR (400 MHz, CDCl3): δ 7.73 (d, J = 2.5 Hz,
1 H), 7.50−7.47 (m, 1 H), 7.33−7.29 (m, 2 H), 7.01 (dd, J = 8.5, 2.0
Hz, 1 H), 4.11 (d, J = 7.5 Hz, 2 H), 3.91 (dd, J = 11.5, 3.5 Hz, 2 H),
3.85−3.79 (m, 4 H), 3.66 (s, 3 H), 3.26 (td, J = 11.5, 2.0 Hz, 2 H),
3.22−3.17 (m, 4 H), 2.25 (s, 3 H), 2.08 (ttt, J = 11.5, 7.5, 3.5 Hz, 1
H), 1.39−1.23 (m, 4 H); LC−MS (method A) m/z: 471 [(M + H)+];
Rt: 0.73 min; 100% purity.
5-(6-(1,1-Dioxidothiomorpholino)-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-
one (23r). 5-(6-Bromo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-
benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one 22b (100
mg, 0.24 mmol), thiomorpholine 1,1-dioxide (32.5 mg, 0.24 mmol),
NaOtBu (23.1 mg, 0.24 mmol), JohnPhos (9.2 mg, 0.03 mmol), and
Pd2(dba)3 (9.0 mg, 0.01 mmol) were suspended in 1,4-dioxane (2
1,3-Dimethyl-5-(5-(3-(methylsulfonyl)pyrrolidin-1-yl)-1-((tetra-
hydro-2H-pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)pyridin-
2(1H)-one (23o). A stock solution of 5-(5-bromo-1-((tetrahydro-2H-
pyran-4-yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-
2(1H)-one 22a (250 mg, 0.12 mmol) was prepared in 1,4-dioxane (4
mL), and an aliquot (0.8 mL) was added to a vial containing 3-
Z
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
Article
mL). The reaction vessel was sealed and heated in a microwave at 90
°C for 20 min. After cooling the reaction to rt, further NaOtBu (23.1
mg, 0.24 mmol), JohnPhos (9.2 mg, 0.03 mmol), and Pd2(dba)3 (9.0
mg, 0.01 mmol) were added to the reaction vessel. The reaction vessel
was sealed and heated in a microwave at 90 °C for 20 min. After
cooling the reaction to rt, the reaction mixture was loaded onto a 1 g
C18 SPE cartridge (preconditioned with MeCN [3 mL]). The
cartridge was flushed with MeCN (3 mL), and the solvent was
removed under a stream of nitrogen. The sample was dissolved in
DMSO (1 mL) and purified by MDAP (method B). The solvent was
dried under a stream of nitrogen to give the title compound (16 mg,
0.03 mmol, 14% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.07 (d, J
= 2.5 Hz, 1 H), 7.72−7.70 (m, 1 H), 7.48 (d, J = 8.5 Hz, 1 H), 7.26
(d, J = 2.0 Hz, 1 H), 6.99 (dd, J = 8.5, 2.0 Hz, 1 H), 4.24 (d, J = 7.5
Hz, 2 H), 3.81−3.75 (m, 4 H), 3.72 (dd, J = 11.5, 3.5 Hz, 2 H), 3.55
(s, 3 H), 3.23−3.18 (m, 4 H), 3.12 (td, J = 11.5, 2.0 Hz, 2 H), 2.10 (s,
3 H), 1.99−1.86 (m, 1 H), 1.27−1.10 (m, 3 H); LC−MS (method F)
m/z: 471 [(M + H)+]; Rt: 0.52 min; 98% purity.
5-(5-(3-Hydroxypyrrolidin-1-yl)-1-((tetrahydro-2H-pyran-4-yl)-
methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one
(23s). Into a vial were added 5-(5-bromo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-
one 22a (62 mg, 0.15 mmol), pyrrolidin-3-ol (20 mg, 0.23 mmol),
RuPhos Pd G1 precatalyst (12 mg, 0.02 mmol), RuPhos (7.0 mg, 0.02
mmol), and LiHMDS (1 M in THF, 0.36 mL, 0.36 mmol) in 2-
MeTHF (0.6 mL). A stirrer bar was added to the vial which was
capped and stirred at 65 °C for 4 h. Pyrrolidin-3-ol (20 mg, 0.23
mmol), RuPhos Pd G1 precatalyst (12 mg, 0.02 mmol), RuPhos (7.0
mg, 0.02 mmol), and LiHMDS (1 M in THF, 0.36 mL, 0.36 mmol)
were added, and the reaction was heated at 65 °C for 1 h. The sample
was purified by MDAP (method B). The solvent was dried under a
stream of nitrogen to give the title compound (4.9 mg, 0.01 mmol, 7%
yield). 1H NMR (400 MHz, DMSO-d6): δ 8.06 (d, J = 2.5 Hz, 1 H),
7.72−7.69 (m, 1 H), 7.45 (d, J = 8.5 Hz, 1 H), 6.63 (d, J = 2.0 Hz, 1
H), 6.60 (dd, J = 8.5, 2.0 Hz, 1 H), 4.92−4.89 (m, 1 H), 4.45−4.39
(m, 1 H), 4.18 (d, J = 7.5 Hz, 2 H), 3.71 (d, J = 11.5 Hz, 2 H), 3.55
(s, 3 H), 3.46 (dd, J = 10.0, 5.0 Hz, 1 H), 3.40−3.31 (m, 2 H), 3.41−
3.05 (m, 3 H), 2.12−2.03 (m, 4 H), 2.00−1.85 (m, 2 H), 1.26−1.07
(m, 4 H); LC−MS (method F) m/z: 423 [(M + H)+]; Rt: 0.51 min;
100% purity.
5-(6-(3-Hydroxypyrrolidin-1-yl)-1-((tetrahydro-2H-pyran-4-yl)-
methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one
(23t). In a vial, a suspension of 5-(6-bromo-1-((tetrahydro-2H-pyran-
4-yl)methyl)-1H-benzo[d]imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-
one 22b (50 mg, 0.12 mmol), pyrrolidin-3-ol (17 mg, 0.20 mmol),
NaOtBu (25 mg, 0.26 mmol), Pd2(dba)3 (11 mg, 0.01 mmol), and
JohnPhos (8 mg, 0.03 mmol) in 1,4-dioxane (1 mL) was prepared.
The vial was sealed and heated in a microwave at 90 °C for 20 min.
The reaction mixture was allowed to cool and then loaded onto a 1 g
C18 SPE cartridge (preconditioned with MeCN [2 mL]). The
cartridge was flushed with MeCN (2 mL) and MeOH (2 mL). The
washes were combined, and the solvent was removed under a stream
of nitrogen. The sample was dissolved in DMSO (1 mL) and purified
by MDAP (method B). The solvent was removed using a plate dryer
to give the title compound (23 mg, 0.06 mmol, 46% yield). 1H NMR
(400 MHz, DMSO-d6): δ 8.04 (d, J = 2.5 Hz, 1 H), 7.71−7.69 (m, 1
H), 7.39 (d, J = 8.5 Hz, 1 H), 6.57 (d, J = 2.0 Hz, 1 H), 6.53 (dd, J =
8.5, 2.0 Hz, 1 H), 4.94 (d, J = 5.0 Hz, 1 H), 4.47−4.41 (m, 1 H), 4.18
(d, J = 7.5 Hz, 2 H), 3.72 (d, J = 11.5 Hz, 2 H), 3.54 (s, 3 H), 3.49
(dd, J = 10.0, 5.0 Hz, 1 H), 3.44−3.31 (m, 2 H), 3.19−3.07 (m, 3 H),
2.14−2.04 (m, 4 H), 2.00−1.88 (m, 2 H), 1.26−1.09 (m, 4 H); LC−
MS (method F) m/z: 423 [(M + H)+]; Rt: 0.48 min; 100% purity.
5-(1-(1,3-Dimethoxypropan-2-yl)-5-morpholino-1H-benzo[d]-
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (24). 5-(5-Bromo-1-
(1,3-dimethoxypropan-2-yl)-1H-benzo[d]imidazol-2-yl)-1,3-dime-
thylpyridin-2(1H)-one 26 (29.5 g, 70.2 mmol), DavePhos (1.381 g,
3.51 mmol), Pd2(dba)3 (1.285 g, 1.404 mmol), morpholine (12.23
mL, 140 mmol), and 2-MeTHF (150 mL) were combined in a round
bottom flask under nitrogen, and then NaOtBu (2 M in THF, 105
mL, 211 mmol) was added. The mixture was heated at 80 °C for 2 h.
The reaction mixture was diluted with brine (200 mL) and extracted
with EtOAc (2 × 200 mL). The combined organics were dried and
evaporated in vacuo to give a brown gum, which was triturated with
EtOAc (200 mL) and then diluted with ether (100 mL), giving a fine,
beige precipitate. This was collected by filtration and washed with
ether to give the title compound (batch 1, 21.2 g).
The filtrate was evaporated in vacuo to give a brown gum. This was
dissolved in DCM (30 mL) and loaded onto a 340 g silica column.
Elution was carried out using a gradient of 0−10% MeOH in DCM,
and product-containing fractions were evaporated in vacuo. The
resulting solid was triturated with ether (100 mL) and collected by
filtration to give the title compound (batch 2, 6.5 g).
5-(5-Bromo-1-(1,3-dimethoxypropan-2-yl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one 26 (33.2 g, 79.0 mmol),
DavePhos (1.554 g, 3.95 mmol), Pd2(dba)3 (1.447 g, 1.580 mmol),
and 2-MeTHF (150 mL) were combined in a round bottom flask
under nitrogen; then, NaOtBu (2 M in THF, 118 mL, 237 mmol) was
added, and the mixture was heated at 80 °C for 2 h. The reaction
mixture was diluted with brine (200 mL) and extracted with EtOAc
(2 × 200 mL). The combined organics were dried and evaporated in
vacuo to give a brown gum, which was triturated with EtOAc (200
mL) and then diluted with ether (100 mL), giving a fine, beige
precipitate. This was collected by filtration and washed with ether to
give the title compound (batch 3, 31.4 g).
5-(5-Bromo-1-(1,3-dimethoxypropan-2-yl)-1H-benzo[d]imidazol-
2-yl)-1,3-dimethylpyridin-2(1H)-one 26 (20 g, 47.6 mmol), Dave-
Phos (0.936 g, 2.379 mmol), Pd2(dba)3 (0.871 g, 0.952 mmol),
morpholine (8.29 mL, 95 mmol), and 2-MeTHF (150 mL) were
combined in a round bottom flask under nitrogen; then, NaOtBu (2
M in THF, 71.4 mL, 143 mmol) was added, and the mixture was
heated at 80 °C for 2 h. The reaction mixture was diluted with brine
(200 mL) and extracted with EtOAc (2 × 300 mL). The combined
organics were dried and evaporated in vacuo to give a brown gum.
This was combined with batches 1, 2, and 3 and dissolved in DCM
(200 mL). The solution was loaded onto a 750 g silica column and
eluted with EtOAc (5 column volumes) and then a gradient of 0−
25% EtOH in EtOAc (20 column volumes). Product-containing
fractions were evaporated in vacuo to give a pale yellow solid. This
was dissolved in DCM (500 mL), SiliCycle thiourea silica resin (40 g)
was added, and then the mixture was stirred at rt for 1 h. The
suspension was filtered, and the filtrate evaporated in vacuo. The
resulting foam was triturated with ether (300 mL), and the resulting
solid was collected by filtration to give a pale yellow solid (60 g). This
was suspended in EtOAc (400 mL) and heated to reflux with stirring
for 1 h. The mixture was allowed to cool over 2 h and then further
cooled using an ice bath and stirred for 1 h. The suspension was
filtered, and the solid was washed with ether (200 mL) to give the title
compound as an almost colorless solid (49.8 g, 117 mmol, 59% total
yield). mp 174 °C; IR (solid) ν (cm−1): 2908, 1653, 1611, 1189,
1105, 968; 1H NMR (400 MHz, DMSO-d6): δ 8.00 (d, J = 2.5 Hz, 1
H), 7.66−7.64 (m, 1 H), 7.63 (d, J = 9.0 Hz, 1 H), 7.10 (d, J = 2.5
Hz, 1 H), 6.96 (dd, J = 9.0, 2.5 Hz, 1 H), 4.77 (tt, J = 9.0, 4.5 Hz, 1
H), 3.97 (dd, J = 10.5, 9.0 Hz, 2 H), 3.76−3.78 (m, 4 H), 3.75 (dd, J
= 10.5, 4.5 Hz, 2 H), 3.53 (s, 3 H), 3.16 (s, 6 H), 3.10−3.07 (m, 4 H),
2.08 (s, 3 H); 13C NMR (150 MHz, DMSO-d6): δ 161.6, 151.5,
147.4, 144.0, 138.8, 137.3, 128.0, 127.4, 113.4, 112.4, 108.1, 105.1,
69.8, 66.2, 58.3, 56.7, 50.3, 37.4, 16.9; LC−MS (method A) m/z: 427
[(M + H)+]; Rt: 0.81 min; 100% purity; HRMS: [(M + H)+] calcd for
C23H31N4O4, 427.2340; found, 427.2340.
4-Bromo-N-(1,3-dimethoxypropan-2-yl)-2-nitroaniline (25). 4-
Bromo-1-fluoro-2-nitrobenzene (50 g, 227 mmol) and 1,3-dimethox-
ypropan-2-amine (32.5 g, 273 mmol) were dissolved in acetonitrile
(300 mL), and K2CO3 (47.1 g, 341 mmol) was added. The mixture
was stirred at 80 °C for 6 h, then the mixture was allowed to cool and
stood over the weekend at rt. The mixture was diluted with water
(500 mL) and extracted with EtOAc (2 × 500 mL). The organic layer
was washed with water (300 mL) and brine (300 mL), dried, and
evaporated in vacuo to give the title compound as an orange solid (70
g, 220 mmol, 97% yield). 1H NMR (400 MHz, CDCl3): δ 8.35 (d, J =
8.0 Hz, 1 H), 8.31 (d, J = 2.5 Hz, 1 H), 7.47 (dd, J = 9.0, 2.5 Hz, 1
AA
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
Article
H), 6.89 (d, J = 9.0 Hz, 1 H), 3.90−3.81 (m, 1 H), 3.62−3.53 (m, 4
H), 3.41 (s, 6 H); LC−MS (method A) m/z: 319, 321 [(M + H)+];
Rt: 1.25 min; 99% purity.
bromodomain pellet was resuspended in 50 mM 4-(2-hydroxyethyl)-
piperazine-1-ethanesulfonic acid (HEPES) (pH 7.5), 300 mM NaCl,
10 mM imidazole, and 1 μL/mL protease inhibitor cocktail and
extracted from the E. coli cells using sonication. Purification was
carried out using a nickel sepharose high-performance column, and
the proteins were washed and then eluted with a linear gradient of 0−
500 mM imidazole with buffer 50 mM HEPES (pH 7.5), 150 mM
NaCl, and 500 mM imidazole, over 20 column volumes. Final
purification was completed by using a Superdex 200 prep grade size
exclusion column. Purified protein was stored at −80 °C in 20 mM
HEPES (pH 7.5) and 100 mM NaCl. The protein identity was
confirmed by peptide mass fingerprinting, and the predicted
molecular weight was confirmed by mass spectrometry.
BRD4 TR-FRET Assay. All assay components were diluted in a
buffer composition of 50 mM HEPES (pH 7.4), 150 mM NaCl, 5%
glycerol, 1 mM DTT, and 1 mM 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate. The final concentration of
bromodomain protein was 10 nM. An Alexa Fluor 647 derivative of I-
BET762 (27) was used as the competing ligand and was used at a
concentration equal to the Kd. These components were premixed and
5 μL of this reaction mixture was added to wells containing 50 nL of
various concentrations of the test compound or DMSO vehicle (0.5%
DMSO final) in 384-well black low volume microtiter plates and
incubated in the dark for 30 min at rt. The bromodomain protein/
fluorescent ligand interaction was detected using TR-FRET following
5 μL addition of a 1.5 nM europium chelate-labeled anti-6His
antibody in assay buffer. Time-resolved fluorescence (TRF) was then
detected on a TRF laser equipped with a PerkinElmer EnVision
multimode plate reader (excitation = 337 nm; emission 1 = 615 nm;
emission 2 = 665 nm; dual wavelength bias dichroic = 400, 630 nm).
The TR-FRET ratio was calculated using the following equation: ratio
= ((acceptor fluorescence at 665 nm)/(donor fluorescence at 615
nm)) × 1000. TR-FRET ratio data were normalized to a mean of 16
replicates per microtiter plate of both 10 μM I-BET151 4 and 1%
DMSO controls, and IC50 values were determined for each of the
compounds tested by fitting the fluorescence ratio data to a four-
parameter model: y = a + ((b − a)/(1 + (10x/10c)d)) where “a” is the
minimum, “b” is the Hill slope, “c” is the IC50, and “d” is the
maximum.
Cocrystallization and Structure Determination. BRD4 BD1-
dimethylpyridone benzimidazole 24 complex was cocrystallized with
at least 3:1 excess of the compound over protein at 9.0 mg/mL in 120
+ 120 nL sitting drops using a 96-well MRC plate. Crystals were
grown with a well solution of 0.1 M PCTP pH 8.0, 25% v/v PEG1500
at 20 °C and cryoprotected using the well solution with 20% ethylene
glycol prior to flash freezing in liquid nitrogen. Data from a single
crystal were collected at 100 K on I03 at the Diamond Light Source at
Oxford and processed to 1.30 Å using X-ray detector software
(XDS)80 and AIMLESS.81 A molecular replacement solution was
determined using phaser and a previously determined in-house
structure as a starting model. The P212121 cell (α = β = γ = 90°, a =
39.493 Å, b = 49.533 Å, c = 58.2133 Å) has a single molecule in the
ASU. Manual model building was performed using COOT82 and
refined using REFMAC83 within the CCP4 software suite. There was
clear difference density for the ligand in the acetylated lysine binding
site, allowing the ligand to be unambiguously modeled. Statistics for
the data collection and refined co-ordinates are given in the
Supporting Information.
Measurement of LPS-Induced MCP-1 Production from
Human WB. Blood was collected in a tube containing sodium
heparin (10 units/mL). 96-well compound plates containing 1 μL test
sample in 100% DMSO were prepared (two replicates to account for
donor variability). WB (130 μL) was dispensed into each well and
incubated for 30 min at 37 °C under a 5% CO2 atmosphere. LPS (10
μL, Salmonella typhosa) made up in phosphate-buffered saline (PBS;
200 ng/mL final assay concentration) was added to each well of the
compound plates. After further incubation for 18−24 h at 37 °C
under a 5% CO2 atmosphere, 140 μL of PBS was added to each well.
The plates were sealed and centrifuged for 10 min at 1800 rpm at rt.
The cell supernatant (20 μL) was placed in a 96-well meso scale
5-(5-Bromo-1-(1,3-dimethoxypropan-2-yl)-1H-benzo[d]-
imidazol-2-yl)-1,3-dimethylpyridin-2(1H)-one (26). 4-Bromo-N-
(1,3-dimethoxypropan-2-yl)-2-nitroaniline 25 (69 g, 216 mmol) was
dissolved in EtOH (400 mL) with heating, and on cooling, the
starting material crystallized out. 1,5-Dimethyl-6-oxo-1,6-dihydropyr-
idine-3-carbaldehyde (35.9 g, 238 mmol) was added to the
suspension, followed by water (200 mL) and Na2S2O4 (94 g, 540
mmol), and the mixture was heated at 90 °C for 18 h. The mixture
was evaporated to approximately half its original volume, then diluted
with water (200 mL), and extracted with DCM (2 × 300 mL). The
combined organics were washed with brine and then dried and
evaporated in vacuo. The resulting yellow solid was triturated with
EtOAc (200 mL), and the solid was collected by filtration. The solid
was washed with ether (200 mL) and dried under vacuum to give the
1
title compound (batch 1, 31 g, 34% yield). H NMR (400 MHz,
CDCl3): δ 7.90 (d, J = 2.0 Hz, 1 H), 7.87 (d, J = 2.0 Hz, 1 H), 7.68−
7.66 (m, 1 H), 7.44 (d, J = 9.0 Hz, 1 H), 7.35 (dd, J = 9.0, 2.0 Hz, 1
H), 4.84 (tt, J = 8.0, 5.0 Hz, 1 H), 3.94 (dd, J = 10.0, 8.0 Hz, 2 H),
3.82 (dd, J = 10.0, 5.0 Hz, 2 H), 3.62 (s, 3 H), 3.28 (s, 6 H), 2.22 (s, 3
H); LC−MS (method A) m/z: 420, 422 [(M + H)+]; Rt: 1.00 min;
98% purity.
The filtrate was evaporated in vacuo, and the residue was dissolved
in DCM (100 mL). The solution was loaded onto a 750 g silica
column, and elution was carried out using a gradient of 0−10%
MeOH in DCM. Product-containing fractions were evaporated in
vacuo to give a yellow solid. This was dissolved in hot EtOAc (150
mL), allowed to cool, and left to stand overnight. The resulting solid
was collected by filtration; the product was washed with ether (2 ×
100 mL), stirring the product/ether mixture to ensure good
trituration, and the product was then dried to give the title compound
(batch 2, 20 g, 47.6 mmol, 22% yield). 1H NMR (400 MHz, DMSO-
d6): δ 8.06 (d, J = 2.0 Hz, 1 H), 7.82 (d, J = 2.0 Hz, 1 H), 7.80 (d, J =
9.0 Hz, 1 H), 7.68−7.66 (m, 1 H), 7.35 (dd, J = 9.0, 2.0 Hz, 1 H),
4.86 (tt, J = 9.0, 4.5 Hz, 1 H), 4.00 (dd, J = 10.5, 9.0 Hz, 2 H), 3.76
(dd, J = 10.5, 4.5 Hz, 2 H), 3.16 (s, 6 H), 2.09 (s, 3 H); LC−MS
(method A) m/z: 420, 422 [(M + H)+]; Rt: 1.00 min; 100% purity.
4-((Z)-3-(6-{[5-(2-[(S)-6-(4-Chlorophenyl)-8-methoxy-1-methyl-
4H-benzo[f ][1,2,4]triazolo[4,3-a][1,4]diazepin-4-yl]acetamido)-
pentyl]amino}-6-oxohexyl)-2-{(2E,4E)-5-[3,3-dimethyl-5-sulfo-1-(4-
sulfobutyl)-3H-indol-1-ium-2-yl]penta-2,4-dien-1-ylidene}-3-meth-
yl-5-sulfoindolin-1-yl)butane-1-sulfonate (27). To a solution of N-
(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-8-methoxy-1-methyl-
4H-benzo[f ][1,2,4]triazolo[4,3-a][1,4]diazepin-4-yl]acetamide58 (1.7
mg, 3.53 μmol) in DMF (40 μL) was added a solution of Alexa Fluor
647-ONSu (2.16 mg, 1.966 μmol) in DMF (100 μL). The mixture
was basified with DIPEA (1 μL, 5.73 μmol) and agitated overnight on
a vortex mixer. The reaction mixture was evaporated to dryness. The
solid was dissolved in a mixture of acetonitrile/water/acetic acid
(5:4:1, <1 mL), filtered, and applied to a Phenomenex Jupiter C18
preparative column. Gradient elution was carried out at a flow rate of
10 mL/min using 0.1% TFA in water (mobile phase A) and 0.1%
TFA in acetonitrile/water (9:1) (mobile phase B) 0−10 min 5%
mobile phase B, 10−100 min 35% mobile phase B, 100−115 min
100% mobile phase B. The appropriate fractions were combined,
evaporated to dryness, and triturated with dry ether, and the blue
solid was dried overnight at <0.2 mbar to give the title compound
(1.54 mg). HPLC analysis was conducted on a Spherisorb ODS2
column. Gradient elution was carried out using 0.1% TFA in water
(mobile phase A) and 0.1% TFA in acetonitrile/water (9:1) (mobile
phase B) 0−60 min 1−35% mobile phase B. Estimated purity >95%.
Mass analysis was recorded on a Waters micromass ZQ mass
spectrometer operating in the positive electrospray ionization mode
giving ES+ = 661.8 corresponding to [(M − C2H5/2) + H]+.
BRD4 Protein Expression and Purification. Recombinant
human bromodomains BRD4 (1−477) (Y390A) (BD2 mutation to
monitor binding to BD1) were expressed in Escherichia coli cells using
a pET15b vector with a 6-His tag at the N-terminal. The His-tagged
AB
DOI: 10.1021/acs.jmedchem.9b01670
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
discovery (MSD) plate precoated with a human MCP-1 capture
antibody. The plates were sealed and placed on a shaker at 600 rpm
for 1.5 h at rt. Anti-human MCP-1 antibody (20 μL) labeled with
sulfo-TAG was added to each well of the MSD plate (stock 50× was
diluted 1:50 with diluent 100, final assay concentration was 1 μg/
mL). The plates were then resealed and shaken for 1.5 h at rt before
washing with PBS Tween 0.05% (×3). 2× MSD Read Buffer T (150
μL, stock 4× MSD Read Buffer T was diluted 50:50 with deionized
water) was then added to each well and the plates read on the MSD
Sector Imager 6000. Concentration response curves for each
compound were generated from the data and an IC50 value was
calculated. Human biological samples were sourced ethically and their
research use was in accord with the terms of the informed consents
under an Institutional Review Board (IRB)/Ethics Committee (EC)-
approved protocol.
administration of dimethylpyridone benzimidazole 24 (in 1% (w/v)
methylcellulose, aq 400) either QD or QOD over a 14 day dosing
period. On day 1 of the study, each mouse received a single bolus ip
administration of TNP-KLH (100 μg/kg) 0.5 h after oral
administration of the compound. Serial blood samples were collected
at 0.5 h post oral compound administration via tail vein on days 1, 4,
7, 9, and 11 or via cardiac puncture (terminal sample) on day 14, and
the serum harvested from the blood samples was frozen at −80 °C.
On the day of analysis, the serum was thawed to rt and levels of IgG1
were measured using a TNP ELISA and the plates read on a
SpectraMax 190 spectrophotometer. Cav values were generated using
WinNonlin Phoenix version 6.3. All values statistically analyzed are
represented as mean SD and are compared to the corresponding
vehicle-treated group. Levels of significance were calculated by
ANOVA followed by Dunnett’s multiple comparison t-test using
GraphPad Prism version 5.04 (GraphPad Software, San Diego, CA).
Statistical differences were determined as p < 0.01 (**) as compared
to the vehicle-treated group. Animal studies were ethically reviewed
and carried out in accordance with Animals (Scientific Procedures)
Act 1986 and the GSK Policy on the Care, Welfare and Treatment of
Laboratory Animals.
Measurement of LPS Induced IL-6 Production from Human
WB. Blood was collected in a tube containing sodium heparin (10
units of heparin/mL of blood). 96-well compound plates containing 1
μL of the test sample in 100% DMSO were prepared (two replicates
to account for donor variability). WB (130 μL) was dispensed into
each well of the 96-well compound plates and incubated for 30 min at
37 °C under a 5% CO2 atmosphere. LPS (10 μL, S. typhosa) made up
in PBS with 1% bovine serum albumin (200 ng/mL final assay
concentration) was added to each well of the compound plates. The
plates were then placed in the humidified primary cell incubator for
18−24 h at 37 °C under a 5% CO2 atmosphere. PBS (140 μL) was
added to all wells of the compound plates containing blood, and the
plates were sealed and shaken on a plate shaker at 600 rpm for 2 min.
The plates were then centrifuged for 10 min at 2500 rpm at rt. The
cell supernatant (100 μL) was removed using a Bomec NX robot.
MSD plates precoated with the human IL-6 capture antibody were
blocked with MSD diluent 2 for 30 min on a plate shaker (600 rpm at
rt). The supernatants were diluted 1:40 in PBS, and 25 μL of which
was added to 96-well MSD IL-6 plates. The plates were sealed and
placed on a shaker at 600 rpm for 1.5 h at rt. Anti-human IL-6
antibody (20 μL) labeled with sulfo-TAG was added to each well of
the MSD plate (stock 50× was diluted 1:50 with diluent 3, final assay
concentration is 1 μg/mL). The plates were then resealed and shaken
for 1 h at rt before washing with PBS Tween 0.05% (×3). 2× MSD
Read Buffer T (150 μL, stock 4× MSD Read Buffer T was diluted
50:50 with deionized water) was then added to each well, and the
plates were read on the MSD Sector Imager 6000. Concentration
response curves for each compound were generated from the data,
and an IC50 value was calculated. Human biological samples were
sourced ethically, and their research use was in accord with the terms
of the informed consents under an IRB/EC-approved protocol.
Mouse LPS Model. Male CD1 mice (Charles River Laboratories,
5 per group) received an iv challenge of LPS (100 μg/kg, L3192 E.
coli 0127:B8) 0.5 h after oral administration of dimethylpyridone
benzimidazole 24 (in 1% (w/v) methylcellulose, aq 400). Serial blood
samples were collected via tail vein up to 3 h or via cardiac puncture
at 5 h (terminal sample) post LPS administration, and the serum
harvested from the blood samples was frozen at −80 °C. On the day
of analysis, the serum was thawed to rt and diluted 1:50 with assay
diluent and levels of IL-6 were measured on MSD plates according to
the manufacturer’s guidelines. Plates were read on an MSD Sector
Imager 6000. The mean IL-6 AUC and mean unbound blood AUC
values were generated using WinNonlin Phoenix version 6.3. All
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01670.
Affinity screening of the encoded library method, BRD4
FP assay method, representative compound spectra, full
assay data table, representative BCP selectivity data for
dimethylpyridone benzimidazole 20a, chromLogD7.4
method, solubility methods and data, X-ray powder
diffraction (XRPD) method and data, mouse in vivo
pharmacokinetic data for 2,6-dimethylphenol benzimi-
dazole 10a, dose prediction calculations, in vitro
clearance data for 5-dimethylamine 22c and 5-morpho-
line 23e, bromodomain binding assay panel data,
NanoBRET method and data, X-ray crystallization
methods and data, and developability and cross
screening panel data (PDF)
Molecular formula strings and biological data for final
compounds (CSV)
Accession Codes
Cocrystal structures of BRD4 BD1 in the complex with
compounds 10, 20a, and 24 and cocrystal structures of BRD2
BD1 in the complex with compounds 16 and 17 are deposited
under the accession codes 6TPX, 6TPY, 6TPZ, 6TQ1, and
6TQ2, in the Protein Data Bank, respectively. Authors will
release the atomic coordinates upon article publication.
AUTHOR INFORMATION
Corresponding Author
*E-mail: christopher.x.wellaway@gsk.com.
■
values statistically analyzed are represented as mean
SD and are
ORCID
compared to the corresponding vehicle-treated group. Levels of
significance were calculated by analysis of variance (ANOVA)
followed by Dunnett’s multiple comparison t-test using GraphPad
Prism version 5.04 (GraphPad Software, San Diego, CA). Statistical
differences were determined as p < 0.05 (*), p < 0.001 (***) as
compared to the vehicle-treated group. Animal studies were ethically
reviewed and carried out in accordance with Animals (Scientific
Procedures) Act 1986 and the GSK Policy on the Care, Welfare and
Treatment of Laboratory Animals.
Christopher R. Wellaway: 0000-0003-0077-3452
Paul Bamborough: 0000-0001-9479-2894
David J. Hirst: 0000-0001-9969-2104
Philip G. Humphreys: 0000-0002-8614-7155
Rishi R. Shah: 0000-0002-5018-1730
Present Address
§Research and Early Development, Respiratory, Inflammation
and Autoimmunity (RIA) BioPharmaceuticals R&D, AstraZe-
T Cell-Dependent Immunization Model. Male CD1 mice
(Charles River Laboratories, 8 per group) received a single oral
̈
neca, SE-431 83 Molndal, Sweden.
AC
DOI: 10.1021/acs.jmedchem.9b01670
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Journal of Medicinal Chemistry
Article
Suto, R. K.; Wong, N. C. W.; Wagner, G. S.; Hansen, H. C.; Young, P.
R. RVX-208, an inducer of ApoA-I in humans, is a BET bromodomain
antagonist. PLoS One 2013, 8, No. e83190.
(7) Picaud, S.; Wells, C.; Felletar, I.; Brotherton, D.; Martin, S.;
Savitsky, P.; Diez-Dacal, B.; Philpott, M.; Bountra, C.; Lingard, H.;
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
Fedorov, O.; Muller, S.; Brennan, P. E.; Knapp, S.; Filippakopoulos, P.
̈
The authors declare no competing financial interest.
RVX-208, an inhibitor of BET transcriptional regulators with
selectivity for the second bromodomain. Proc. Natl. Acad. Sci. U.S.A.
2013, 110, 19754−19759.
ACKNOWLEDGMENTS
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Zeng, L.; Kaur, J.; Moy, G.; Rusinova, E.; Rodriguez, Y.; Matikainen,
B.; Vincek, A.; Joshua, J.; Casaccia, P.; Zhou, M.-M. Selective
chemical modulation of gene transcription favors oligodendrocyte
lineage progression. Chem. Biol. 2014, 21, 841−854.
We are grateful to Graham Inglis, Emmanuel Demont, and
Cesar Molina for review and proof-reading of the manuscript.
We thank Kate Dennis, Alan Ferrie, Iain Reid, Yang Zhuli and
colleagues at WuXi, and GVK for supporting laboratory work.
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ABBREVIATIONS
■
BAZ2A, bromodomain adjacent to zinc finger domain 2A;
BCP, bromodomain-containing proteins; BD1, first (N-
terminal) bromodomain; BD2, second (C-terminal) bromo-
domain; BET, bromodomain and extraterminal; BOP,
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexa-
fluorophosphate; BRET, bioluminescence resonance energy
transfer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-
1-propanesulfonate; dba, dibenzylideneacetone; DEL, DNA-
encoded small-molecule library; DIPEA, diisopropylethyl-
amine; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpro-
pane-1,3-diamine; ELT, encoded library technology; HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate; HEPES, 4-(2-
hydroxyethyl) piperazine-1-ethanesulfonic acid; Heps, hepato-
cytes; HOBt, 1H-benzo[d][1,2,3]triazol-1-ol; HPB, hydrox-
ypropyl betacyclodextrin; IL-6, interleukin 6; IVC, in vitro
clearance; KAc, N-acetyl lysine; MCP-1, monocyte chemo-
attractant protein 1; MDAP, mass-directed autopreparative
high-performance liquid chromatography; 2-MeTHF, 2-methyl
tetrahydrofuran; Mics, microsomes; MMP, matched molecular
pair; NMC, nuclear protein in testis midline carcinoma; PFI,
property forecast index; PKPD, pharmacokinetic/pharmacody-
namic; PPA, polyphosphoric acid; PROTAC, proteolysis
targeting chimera; PTM, post-translational modification;
TNP-KLH, trinitrophenyl-keyhole limpet hemocyanin; TRF,
time-resolved fluorescence; WB, whole blood; XRPD, X-ray
powder diffraction
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chemical probes by structure-guided medicinal chemistry and
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