Template-Hopping Approach Leads to Potent, Selective, and Highly Soluble Bromo and Extraterminal Domain (BET) Second Bromodomain (BD2) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c02156

A number of reports have recently been published describing the discovery and optimization of bromo and extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 (3) and GSK620 (5). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds 28 (GSK452), 39 (GSK737), and 36 (GSK217).

Full text of DOI:10.1021/acs.jmedchem.0c02156

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Article
Template-Hopping Approach Leads to Potent, Selective, and Highly
Soluble Bromo and Extraterminal Domain (BET) Second
Bromodomain (BD2) Inhibitors
Helen E. Aylott,* Stephen J. Atkinson, Paul Bamborough, Anna Bassil, Chun-wa Chung, Laurie Gordon,
Paola Grandi, James R. J. Gray, Lee A. Harrison, Thomas G. Hayhow, Cassie Messenger,
Darren Mitchell, Alexander Phillipou, Alex Preston, Rab K. Prinjha, Francesco Rianjongdee,
Inmaculada Rioja, Jonathan T. Seal, Ian D. Wall, Robert J. Watson, James M. Woolven,
and Emmanuel H. Demont
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ABSTRACT: A number of reports have recently been published describing the discovery and optimization of bromo and
extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 (3)
and GSK620 (5). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide
functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-
based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore
the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds 28 (GSK452), 39 (GSK737), and 36
(GSK217).
pan-BET inhibition;25,27−29 hence, some efforts in this target
INTRODUCTION
■
area have focused on enabling selectivity within the BET family
The bromo and extraterminal domain (BET) family is
in attempts to tease apart efficacy and pharmacology-driven
composed of the germ cell-specific (BRDT) and ubiquitously
toxicity.30−38 Additionally, recent studies have shown that the
expressed (BRD2, BRD3, and BRD4) epigenetic reader
BD1 and BD2 domains have different roles in chromatin
proteins. All family members contain N-terminal tandem
binding.39−42 A number of publications have reported
bromodomains, a structural feature that enables the recog-
medicinal chemistry efforts toward the identification of
nition and binding to acetylated lysine residues on histones
molecules with increased selectivity within the BET family.
and other cellular proteins that support their function as key
Due to the higher homology between the four BD1
transcriptional regulators.1 The therapeutic potential of “pan-
bromodomains and the four BD2 bromodomains compared
BET” inhibitors (binding equipotently to all eight bromodo-
mains of the four BET proteins) has been comprehensively
reported for oncology2−11 and immunoinflammation.12−23 The
Received: December 14, 2020
Published: March 4, 2021
potential of this epigenetic reader target family is further
bolstered by the fact that several of these inhibitors are
currently being progressed in the clinic for oncology
indications.24−26 However, it is well-documented that clinical
toxicities and dose-limiting findings have been associated with
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Figure 1. Structures of BD2-biased inhibitor RVX-208 and BD2-selective inhibitor ABBV-744 and their reported affinities to the BD1 and BD2
bromodomain of BRD4. Related amide substituent to those in Table 1 is shown in blue.
a
Table 1. Structure and Profile of 3 (GSK046), Pyridyl 4 (GSK941), and 5 (GSK620)
compound
3 (GSK046)
4 (GSK941)
5 (GSK620)
b
BRD4 BD1/BD2 pIC50 (n)
4.2 (10)/7.3 (14)
1000
0.33/0.38
1.3/414
2.8/4.8
<4.3 (3) /6.8 (4)
315
4.8 (14)/7.1 (16)
200
0.41/0.51
0.1/325
3.1/5.1
BRD4 BD2 selectivity (fold)
c
BRD4 BD2 LE/LLEAT
0.42/0.37
2.5/297
3.7/5.7
≥102/310
390
clogP/MW
chromLogD7.4/PFI
d
e
CLND (μg/mL)/FaSSIF (μg/mL)
91/996
17
≥154/25
200
f
AMPpH=7.4 permeability (nm/s)
a
Related amide substituents are shown in blue. Compounds 3 and 5 are now available from the Structural Genomic Consortium (SGC), visit:
https://www.sgc-ffm.uni-frankfurt.de/. Also tested 4.9 (n = 1). Ligand lipophilicity index.66 CLND = chemiluminescent nitrogen detection
measured at pH = 7.4. FaSSIF = fasted-state simulated solubility measured at pH = 6.5. AMP = artificial membrane permeability measured at pH
b
c
d
e
f
= 7.4.
to the two bromodomains of any given BET protein, these
inhibitors show some degree of selectivity either for the four
BD1 or four BD2 domains. The first generation of pan-
BD1,43−48 BRD4 BD1,49,50 and pan-BD251−53 inhibitors,
including the clinical asset RVX-208 (1, Figure 1) showed
limited degrees of selectivity (<100 fold) and it is only recently
that molecules with a high degree of BD154−56 or BD257
selectivity have been identified, as exemplified with the highly
BD2-selective inhibitor ABBV-74458,59 (2, Figure 1) currently
in phase I oncology trials.
We have previously reported our own effort toward the
identification of drug-like BD2-selective inhibitors60,61 and, in
particular, the identification of 3,62 4,63 and 564 (GSK046,
GSK941, and GSK620, respectively, Table 1). In a similar way
to ABBV-744, they all contain related amide substituents (blue
substructures in Figure 1 and Table 1). This functionality
contributes to selectivity for the BD2 bromodomains over the
BD1s by virtue of its interactions with a histidine residue in the
BD2s (H433 in BRD2), which is replaced by aspartic acid in
the BD1s.58,59,62−64 Throughout this report, BRD4 potency is
used as a representative of BET potencysome BRD2, 3, and
T data can be found in Table 8. Inhibitor 3 showed the highest
potency and selectivity for BRD4 BD2 among our inhibitors
and has favorable physicochemical properties, with a
chromLogD of 2.8, a property forecast index (PFI) of 4.8,65
and reasonable solubility as measured by kinetic solubility
(chemiluminescent nitrogen detection, CLND), with a value
slightly below the acceptable benchmark of 100 μg/mL, but
high solubility in the more relevant fasted-state simulated
intestinal fluid (FaSSIF) solubility, with FaSSIF solubility
above the acceptable benchmark value of 100 μg/mL. Its
permeability measured by an artificial membrane permeability
(AMP) was below the acceptable benchmark value of 50 nm/s,
likely due to the presence of three hydrogen-bond donors.
Compound 3 also contained an embedded aniline which
ultimately halted its progression due to the associated
genotoxic risk.
Herein, we describe our approach to mitigating this
genotoxic risk through a template-hopping and hybridization
approach: first, an isosteric replacement of the acetamide-
acetylated lysine (AcK) mimetic of 3 with a heterocycle was
sought, and second, the learnings from a second BD2-selective
series were incorporated to guide the required redesign of the
core.
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Table 2. Comparison of Different Five-Membered Heterocycles (6−10) against Original Acetamide 11
a
b
c
Also tested <4.3 (n = 1) and <4.8 (n = 1). Also tested 4.4 (n = 1). Also tested 5.0 (n = 1).
core to enable more distant interactions with the bromodo-
main to be sustained.
Table 3. Structure and Profile of Initial Leads 13 and 14
A range of five-membered heterocycles (6−10), which
contained the appropriate methyl group and ortho-heteroatom
arrangement inferred from Figure 2a, were investigated and
evaluated by their biochemical and physicochemical data
(Table 2). All heterocycles showed an increase in BD2 potency
compared with acetamide fragment 11. The ligand efficiency of
8−10 was comparable to that of the acetamide; however, the
isoxazole (6) and the C-linked 1,2,3-triazole (7) showed ligand
efficiencies greater than that of the acetamide. The cyclo-
hexanol amide of compound 3 was omitted in order to identify
the best AcK mimetic before investigating substituents, so it
was unsurprising that these compounds show little selectivity
over BD1. The 1,2,3-triazole AcK mimetic was selected for
further exploration over the isoxazole due to more favorable
lipophilicity (chromLogD measurement of 5.2 compared with
7.2 for the isoxazole), which was reflected in the superior
kinetic solubility shown for 7.
A crystal structure of BRD2 BD2 bound to 1,2,4-triazole 10
was obtained and overlaid with that of 3, as shown in Figure
2b. Despite a specific NH interaction with the protein, we
hoped that triazole 10 was a fair representation of how the
other, preferred 1,2,3-triazole 7 would bind. This structure
confirmed the excellent overlay of the AcK mimetics of the two
compounds with N1 of the triazole making the key hydrogen
bond to N429. The core phenyl group of 10 is pushed further
from the AcK binding pocket by the larger heteroaryl AcK
mimetic. Despite this change, the benzyloxy group maintains a
similar interaction with the WPF region of the shelf to 3. This
is an important region where positioning a lipophilic
substituent close to W370 can lead to increased small molecule
potency (Figure 2b).
compound
13
14
BRD4 BD1/BD2 pIC50 (n)
selectivity (fold)
BD2 LE/LLEAT
cLogP/ChromLogD7.4
CLND (μg/mL)
4.9 (5)/6.9 (5)
100
0.38/0.36
2.3/4.1
46
4.7 (6)/7.0 (6)
170
0.40/0.36
2.5/3.7
≥157
For the latter, the starting point was pyridyl amide 4 (Table
1), whose amide AcK mimetic is reversed in direction
compared to that of 3.63 Compound 4 was an early second-
generation lead originating from the selective probes that have
been previously introduced (e.g., 5, GSK620,64 Table 1),
showing reasonable potency and selectivity for BD2 alongside
acceptable physicochemical properties with a chromLogD of
3.7 and high solubility and permeability.
RESULTS AND DISCUSSION
■
Prior to consideration of the pyridyl core, attention was
focused on a suitable heterocyclic replacement for the
acetamide of 3. Previous research on BET inhibitors has
shown that a number of heterocycles can be used as suitable
AcK mimetics,67−69 not least iBET762 and (+)-JQ1.6,70 By
comparison of the published crystal structures in BRD4 BD1 of
(+)-JQ1 and in BRD2 BD2 of 3 (Figure 2a), it was observed
that the carbonyl oxygen of 3 and one of the triazole N atoms
overlaid closely, each making similar interactions with the
conserved asparagine side chain. A movement of the core was
anticipated following the introduction of the heteroaryl AcK
mimetics that would likely result in a need to reoptimize the
Previous work on the series which led to compound 3
showed that incorporation of the cyclohexanol amide in the
para-position led to an increase in potency and selectivity, due
to a key, water-bridged hydrogen bond between the amide NH
and N429 (BRD2 BD2 numbering).62 This is in slight contrast
with the series’ which led to compounds 5 (GSK620) and 12
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Table 4. Structure and Profile of Initial Leads 13 and 14 Compared with α-Methyl Analogues 17 and 18
a
Racemic material.
where the amide NH forms a direct hydrogen bond with N429
(Figure 2c).63,64 From the crystal structure of 10, it was clear
that a para-amide substituent would not be able to engage
N429. However, it suggested that moving the amide to the
meta-position on the heterocycle-containing molecule could
provide a viable vector for obtaining this interaction.
As has been mentioned previously, the 1,2,3-triazole 7 was
selected for further exploration due to its favorable lipophilicity
and BD2 potency (Table 2). Incorporation of a simple amide
group onto fragment 7 gave ethyl amide 13 and led to a
significant increase in BD2 potency and selectivity over BD1
(Table 3). This also resulted in a drop in clogP (2.8−2.3) and
chromLogD (5.2−3.9) but did not lead to an increase in
solubility (Table 3).
Before embarking on the optimization of this promising
starting point, we decided to also evaluate the potential of
utilizing the alternative core and substitution pattern of pyridyl
4 (Table 1) to maximize the opportunity to identify drug-like
inhibitors bearing a heteroaryl AcK mimetic. In Figure 2c, an
overlay of a representative example of this series for which we
had a crystal structure, pyridyl 12, with triazole 10 is shown.
The core phenyl ring of 10 and the core pyridine ring of 12
overlay approximately. This suggested that it could be possible
to replace the core of 10 with those of 12 and retain the
favorable interactions made by its substituents. Hence, the
pyridyl series and 1,2,3-triazole template were “hybridized” to
give 14 (Table 3).
WPF shelf and amide substituents, their binding modes are
representative of those of these two series of inhibitors. A good
overlay is achieved in the AcK mimetic-core region, and a very
similar access to the amide vector region is seen for both
templates. However, the access to the WPF shelf is dissimilar,
with the aromatic rings sitting in different orientations that do
not overlay. Both aromatic rings form aromatic interactions
with W370 and H433; in the case of the benzyloxy series
analogue 15, both of these interactions are edge-to-face, while
pyridyl series analogue 16 shows an edge-to-face interaction
with H433 but an offset stacking interaction with W370. This
can be explained by the differences in the vectors of the linkers
between the cores and WPF shelf groups, which cause the rings
to sit in different orientations. Therefore, we thought that this
may lead to differences in structure−activity relationship
(SAR) between the two series in the WPF shelf region. These
differences are exemplified below.
In the original pyridyl and acetamide series (4 and 3 as
representative examples, Figure 1), adding an α-methyl to the
benzylic position gave an increase in potency, often around
threefold, due to the methyl group making an extra lipophilic
interaction in the ZA channel region, as exemplified by 3.62,63
This vacant region is also shown in Figure 3a and it seemed
reasonable to investigate if productive interactions could be
made with these heteroaryl series.
Incorporation of the methyl group gave contrasting results
(Table 4). For the benzyloxy derivatives, we saw the expected
boost in potency with 17 being ∼threefold more potent than
13. However, for the pyridyl series, no increase in potency was
observed. The protein crystallography offers a possible
rationale for these findings, in that the benzylic vector in the
unsubstituted pyridyl template is not directed toward the ZA
channel (Figure 3a). As a result, the inhibitor is shown to
subtly change conformation with the addition of a methyl
group (18, Figure 4), which causes the amide vector to adopt a
less-preferred conformation. This is not the case for benzyloxy
Compound 14 showed comparable potency and selectivity
with 13 but had increased ligand efficiency, although ligand
efficiencies were high for both compounds. The two
compounds had similar lipophilicity, although 14 had higher
solubility.
The binding mode of the two templates once functionalized
with an amide is shown in Figure 3, using phenyl derivative 15
and pyridyl 16 for which we managed to obtain crystal
structures in BRD2 BD2. If these two compounds have specific
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Table 5. WPF Shelf SAR Exploration for Benzyloxy Series
a
b
c
d
Also tested 4.3 (n = 1) and 4.8 (n = 1). Also tested <4.3 (n = 2). Also tested <4.3 (n = 1). Also tested 4.4 (n = 1) and 5.3 (n = 1).
17 (Figure 4) and hence the increase in potency is only
observed for this template.
group and the phenyl core. The biochemical data showed that
while this extended linker was tolerated, it led to a >10-fold
drop in potency; hence, all other analogues were made with the
identical length of 13.
The opposite enantiomer of the original α-Me benzyl
analogue 17 was synthesized to give 21. Protein crystallog-
raphy of 17 suggested that the (S)-enantiomer would be
preferred over the (R)-enantiomer because it projected toward
the ZA channel (Figure 4). This was reflected in the BRD4
data where (S)-compound 17 showed significantly higher
potency and selectivity for BD2 than the (R)-analogue 21. In
Following this initial work, further investigation was carried
out into the WPF shelf SAR on the two templates. These data
were divergent given the different trajectories, the templates
accessed the WPF shelf and the data will be discussed
independently.
For the benzyloxy series, incorporation of heteroatoms into
the phenyl ring, as well as small additional substituents, was
evaluated (Table 5). Compound 19 was synthesized to
investigate the optimal linker length between the WPF shelf
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Table 6. WPF Shelf SAR Exploration for Pyridyl Series
addition, the incorporation of the α-Me group in 17 led to an
increase in kinetic solubility in comparison with the parent
analogue 13.
Next, saturation of the ring was investigated using a THP
group (20), which led to an almost 100-fold drop in potency in
comparison with the parent phenyl (13). Therefore, efforts
were focused on investigating SAR through other aromatic ring
replacements.
Nitrogen-containing heterocycle-based analogues were
synthesized, and all pyridine isomers (15, 22−23) and a
pyrimidine (24) showed measurable potencies, with the 2-
pyridine 15 offering the best potency and selectivity. This was
contrary to other BD2-selective series where a pyridine on the
WPF shelf led to a significant loss of potency;63,64 however, it
was clear from protein crystallography that this template
occupied the WPF shelf differently to other series. The 2-
pyridine 15 also had comparable ligand efficiency with parent
13 and showed an increase in LLEAT by comparison. As
expected, these compounds were less lipophilic than the parent
phenyl and also had higher solubility. The drop in lipophilicity
may also explain the decreases observed in membrane
permeability, although the pyrimidine had much lower AMP
than the pyridines despite having a similar chromLogD7.4.
Based on previous SAR on the acetamide template, electron-
donating methoxy-substituted phenyls 25−27 were also tested:
the ortho-methoxy analogue 25 showed the highest potency
and selectivity of the three analogues, although it showed no
advantage over the parent phenyl 13. All three methoxy-
substituted compounds showed comparable permeabilities
with 13 but large increases in solubility despite comparable
chromLogD values. Finally, the 2-pyridine and α-Me were
combined to give 29 which gave a good balance of key
properties: high potency, selectivity, and ligand efficiencies
with a reasonable chromLogD, solubility, and permeability.
This group was therefore selected to explore the SAR for the
amide vector (vide infra).
A similar approach to SAR exploration of the WPF shelf was
taken with the pyridyl template (Table 6). Unlike the
benzyloxy analogues, pyridine isomers were not synthesized
in this template due to previous data from the parent series
showing that they were not tolerated.63 Since the WPF shelf
groups of this template, such as 4 as a representative example,
and compounds such as 16 overlaid well, this was considered a
reasonable approach.
On this template, electron-rich and electron-poor aromatic
rings were investigated using methoxy- and fluoro-substituted
phenyls (30−35, Table 6). Similar to the benzyloxy series, the
ortho-methoxy 30 gave the best potency and selectivity relative
to the meta- and para-substituted analogues 31 and 32, while
the three fluoro-substituted rings (33−35) were equipotent
and selective; however, none of these six compounds offered a
potency or selectivity advantage over the parent phenyl 14.
With the exception of the meta-methoxy analogue 31, both of
these strategies led to compounds with decreased solubility in
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disadvantageous for ligand efficiency so were not investigated
further.
Table 7. Amide SAR Exploration for Benzyloxy and Pyridyl
Series
Finally, a bicyclic ring was investigated in the form of indole
36. This group had led to significant increases in potency and
selectivity in the original pyridone series.64 Here, a ∼threefold
increase in potency was observed (comparing 36 with 14).
Indole 36 also helped to lower the chromLogD in comparison
with the parent phenyl 14, while maintaining the high
permeability and solubility. This indole compound was also
the most selective and potent compound across the two
templates, up to this point.
The SAR for the amide vector of the two templates was
explored side-by-side due to the similar overlay in the
crystallography of this portion of the molecules. For the
benzyloxy series, the optimum α-Me 2-pyridyl group was used,
while in the pyridyl template, the phenyl was selected over the
indole due to synthetic challenges at the time.
In general, the amide SAR between the two templates was
comparable and tracked with that of the previous series.62−64
The primary amides (37 and 47) caused a drop in potency and
selectivity in comparison with the ethyl amides. While the
methyl amide was not made in the pyridyl template,
incorporation of this group into the benzyloxy series to afford
38 led to comparable potency and selectivity with the ethyl
amide but decreased lipophilicity which in turn reduced
permeability. The cyclopropyl amides 39 and 16 offered no
advantage over the ethyl amides in terms of potency or
physicochemical properties, which is counter to the previous
pyridyl and pyridone templates in which incorporation of this
group led to a boost in potency and selectivity.63,64
A selection of substituents in this area was made based on
SAR from historical templates. Hence, the oxygen-containing
bicycle (40 and 48) was investigated, giving an increase in
potency, particularly for the benzyloxy series, but this came
without an increase in selectivity. Other saturated heterocycles
were also synthesized: the directly linked THPs 41 and 49
appeared to be comparable to the ethyl amide with very similar
biochemical and physicochemical data, while the homologated
THPs 42 and 50 were not as well-tolerated. The cyclohexanol
amide that was used in the acetamide lead compound 3 (Table
1GSK046) was synthesized in the benzyloxy series (43) and
showed reasonable potency but low selectivity for BRD4 BD2,
thus highlighting the differences between the two series.
Finally, a range of extended six-membered ring amines were
installed to provide further highly soluble compounds and
possibly crystalline derivatives, either as the piperidine 54 or as
morpholines 44−45 and 51−52, and fluoro-piperidines 46, 53,
and 55 to modulate the pKa of the basic center. This strategy
not only produced compounds with very high potencies and
selectivities and lower lipophilicities but also led to appreciable
drops in permeability in comparison with the ethyl amide. In
both templates, high kinetic solubility was maintained with the
incorporation of these amides as the measured values were at
the upper limit of the assay for the benzyloxy analogues 44−46
and for the pyridyl analogues 51−55. Nevertheless, fluoropi-
peridine amides 46 and 55 had the highest potencies and
selectivities observed in these two templates, and given that the
upper limit of the BRD4 assay was pIC50 ∼8, it is possible that
these compounds were more potent, and therefore selective,
than quoted here.
a
b
Trans diastereomer. Also tested 4.4 (n = 1).
comparison with the parent phenyl analogue, with little change
to the lipophilicity. Additionally, this strategy offered no
advantage for potency and selectivity in comparison with
parent compound 14, and the six compounds (30−35) were
Chemistry. The SAR for the benzyloxy template was
investigated via synthesis of intermediate 57 (MOM protected
ester), which was prepared from the commercially available
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Table 8. Profile of Lead Compounds 28 (GSK452), 39 (GSK737), and 36 (GSK217)
compound
GSK452, 28
GSK737, 39
7.3 (3)/5.3 (3)
GSK217, 36
7.5 (4)/5.0 (4)
a
BRD4 BD2/BD1 pIC50 (n)
BRD2 BD2/BD1 pIC50 (n)
BRD3 BD2/BD1 pIC50 (n)
BRDT BD2/BD1 pIC50 (n)
BRD4 BD2 selectivity (fold)
BRD4 BD2 LE/LLEAT
6.6 (5)/4.3 (3)
5.8 (3)/<4.3 (4)
6.5 (4)/<4.3 (4)
6.4 (4)/<4.3 (4)
200
0.35/0.39
6.6 (2)/5.6 (2)
1.3/2.7
6.6 (2)/4.4 (2)
7.1 (2)/4.9 (2)
ND
7.3 (2)/4.6 (2)
7.5 (2)/4.5 (2)
7.3 (2)/4.6 (2)
300
0.38/0.36
7.6 (2)/6.4 (2)
2.5/3.1
100
0.37/0.42
7.8 (2)/6.7 (2)
1.1/3.1
PBMC/hWB pIC50 (n) (MCP-1)
cLogP/ChromLogD7.4
AMPpH=7.4 (nm/s)
470
150
300
CLND (μg/mL)/FaSSIF (μg/mL)
hERG pIC50 (n)/has
hepatocyte cli (rat/hu) (mL/min/g tissue)
137/702
<4.3 (3)/84%
1.09/1.18
145/603
4.4 (1) /84%
<0.80/<0.45
148/297
4.5 (1) /90%
b
b
1.98/<0.45
c
rat ivPK (1 mg/kg): CLb (CLb,ub) (mL/min/kg), t1/2 (h); AUC (ng h/mL), 19 (102), 0.45; 873, 0.7, 14 (19), 1.65; 1175, 1.8, 21 (205 ), 0.87, 778; 1.1,
Vss (L/kg), fub
0.19
0.76
nd
d
rat poPK (3 mg/kg): Cmax, tmax; AUC/D (min kg/L), F (%)
634, 0.28; 30, 59
281, 0.25; 23, 32
366, 0.25; 15, 31
a
b
c
d
Also tested <4.3 (n = 2). Also tested <4.3 (n = 2). Calculated using HSA. Values are based on n = 3 mean apart from tmax which is n = 3
median.
Figure 2. X-ray crystal structures (a) (+)-[JQ1] (cyan, PDB: 3MXF6) in BRD4 BD1 overlaid with 3 (GSK046, green, PDB: 6SWP62) in BRD2
BD2; (b) 3 (GSK046, green) and 10 (yellow, PDB: 7NQ5) in BRD2 BD2; (c) 10 (yellow) overlaid with 12 (magenta, PDB: 7NQ9); and (d)
structure of 12.
bromo methylester phenol 56 (Scheme 1). The phenol was
first protected with an MOM group to give intermediate 63.
This underwent Sonogashira cross-coupling with trimethylsi-
lane (TMS)-protected acetylene, giving alkyne 64, and
subsequent 1,3-dipolar cycloaddition with sodium azide and
in situ methylation with methyl iodide gave 1,2,3-triazole 57.71
Route flexibility here allowed either amide or WPF shelf SAR
investigation. For amide SAR evaluation, triazole 57 was first
subjected to acidic conditions to remove the MOM-protecting
group to afford 65. The phenol could then undergo a
Mitsunobu coupling to install the WPF shelf group 59, giving
inversion of stereochemistry as expected. From here, the
intermediate was hydrolyzed to give acid 66. Finally, HATU-
mediated amide coupling gave the fully elaborated final
compounds 13 and 37−43. Where applicable, a Boc
deprotection was then carried out using trifluoroacetic acid
(TFA) to give the free amine final compounds 44−46.
Similarly, for WPF shelf SAR exploration, 1,2,3-triazole
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afford 80 or 81. This reaction was not high-yielding due to the
formation of a byproduct via bis-substitution of the acetylene
unit in a 1:1 ratio with 80−81. Following this, alkyne 80 or 81
was subjected to a 1,3-dipolar cycloaddition using the same
conditions, as in Scheme 1, to give 1,2,3-triazole 69 or 70.71 As
with the previous template, the order of steps could be
switched at this point to allow for SAR investigation of the two
vectors, amide and WPF shelf. For investigation of amide SAR,
the WPF shelf group was installed via a Negishi cross-coupling
to afford 73 or 74. The compound was then hydrolyzed with
TFA or LiOH to give acid 82, which could then undergo
HATU-mediated amide coupling to give the desired final
compounds 14, 16, and 48−50. Where a Boc-protected amine
had been installed into the molecule, this was then removed
using TFA to give final compounds 51−55. Carrying out the
ester hydrolysis and subsequent amide coupling first on
intermediate 69 or 70 allowed for efficient exploration of WPF
shelf SAR from the late-stage intermediate 71 or 72. This
intermediate then underwent Negishi cross-coupling using
either Pd(PPh3)4 or (PPh3)2PdCl2, depending on the pyridyl
halide being used, to give final compounds 14 and 30−35.
For the preparation of indole 36, a different synthetic route
was employed (Scheme 3). Starting with bromomethyl methyl
ester pyridine 83, the Sonogashira cross-coupling and 1,3-
dipolar cycloaddition reactions were carried out using the same
conditions outlined in Scheme 2 to give intermediate 84.71
This was then subjected to oxidation conditions using mCPBA
to give N-oxide species 85, which was converted to an alcohol
via Boekelheide rearrangement with TFAA. A chlorination
with thionyl chloride then gave chloro intermediate 86, which
was used as a handle for an sp2−sp3 Suzuki cross-coupling with
an indole boronic acid using XPhos Pd G1 to give 87.72 This
reaction was low-yielding at 10%; however, optimization was
not carried out as this was considered good enough to provide
the small amount of compound required. Following this, the
methyl ester was hydrolyzed with LiOH to give an acid that
then underwent HATU-mediated amide coupling to give the
fully elaborated compound 36.
Figure 3. (a) X-ray crystal structure of 15 (yellow, PDB: 7NQ8) and
16 (magenta, PDB: 7NQI) in the BRD2 BD2 protein and the shape
complementarity in the binding site; (b) structures of 15 and 16.
As one of the most selective and drug-like compounds made
in the first iteration, 28 (GSK452, Table 5) was progressed
into rat PK studies to provide a benchmark (Table 8).
Following this, the two compounds with the best balance of
potency, selectivity, lipophilicity, and solubility were identified
and further profiled: 36 (GSK217, Table 6) and 39 (GSK737,
Table 7). The three compounds showed the expected pan-
BET BD2 selectivity, with largely equivalent potencies and
selectivities for the BRD2, BRD3, and BRDT BD2 domains
over the BD1 domains as was seen in the BRD4 assay. Here,
analogue 36 (GSK217) particularly maintained high potency
and high selectivity across the four BET bromodomains. The
selectivity profile of this molecule was followed up in the
DiscoverX BromoScan platform of the wider bromodomain
family, and selectivity for the BET BD2 domains was
confirmed (Figure 5).
The three compounds were also evaluated in cellular assays:
an LPS-stimulated human whole blood (hWB) assay and an
LPS-stimulated peripheral blood mononuclear cell (PBMC)
assay.62−64 These assays measure monocyte chemoattractant
protein 1 (MCP-1/CCL2) levels, which is a chemokine that
recruits monocytes, memory T-cells, and dendritic cells to sites
of inflammation. In the PBMC assay, retention of potency
from the biochemical BRD4 BD2 data was observed. In the
hWB assay, we observed a drop-off versus biochemical potency
Figure 4. X-ray crystal structure of 17 (cyan, PDB: 7NQ7) and 18
(orange, PDB: 7NQJ) in the BRD2 BD2 protein, showing the shape
complementarity to their binding site.
intermediate 57 underwent ester hydrolysis and subsequent
HATU-mediated amide coupling to give 58. Following MOM
deprotection, this was then subjected to either Mitsunobu
coupling with the relevant alcohol or nucleophilic substitution
with the relevant bromide to give the final compounds 13, 15,
17, and 19−29.
For the pyridyl series, SAR investigation was enabled using
similar chemistry to the benzyloxy derivatives via key
intermediate 67 or 68. First, the 1,2,3-triazole was installed
from the commercially available dichloro tert-butyl ester
pyridine or dibromo methyl ester pyridine which was subjected
to Sonogashira cross-coupling conditions using Pd(PPh3)4 to
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a
Scheme 1. Alternative Routes for the Preparation of Benzyloxy Analogues
a
Reagents and conditions: (i) MOMCl, Cs2CO3, DMF, 0 °Crt, 16 h (quant.); (ii) TMS acetylene, Et3N, Cu(I)I (10 mol %), (PPh3)2Pd(II)Cl2
(1 mol %), 50 °C, 6 h (76%); (iii) NaN3, MeI, pyridine, K2CO3, vitamin C (40 mol %), Cu(II)SO4 (20 mol %), H2O/MeOH (1:1), rt, 48 h
(79%); (iv) NaOH or LiOH, THF/H2O (1:1), rt50 °C, 3−4 h (60−79%); (v) R2NH2, HATU, DIPEA, DMF, rt, 3−16 h; (vi) HCl, MeOH,
rt50 °C, 3−35 h (80−98%); (vii) ROH, CMBP, DMF, 170 °C, 2−3 h; (viii) RBr, K2CO3, acetone or DMF, rt50 °C, 25−67 h; (ix) TFA,
CHCl3, rt, 1−3 h (14−75%).
for the three compounds, most likely due to protein binding
and the need to permeate into the cell; however, the ranking of
activities between compounds remained the same in both
assays.
All three compounds were in a reasonable lipophilicity
space, and 28 had a slightly lower chromLogD than 39 and 36.
Despite this, 28 had the highest permeability in the AMP assay,
perhaps owing to the fact that the NH of the indole in 36 may
hinder permeation through the lipophilic membrane. Addi-
tionally, the more-relevant FaSSIF solubility was determined,
showing that all three compounds had high FaSSIF solubility
>100 μg/mL. 28, 36, and 39 were tested for off-target toxicity
in the hERG assay, and all three compounds showed no
measurable activity.
To understand the pharmacokinetic properties of these
compounds, they were first evaluated in an in vitro clearance
assay, using rat and human hepatocytes. All three compounds
showed moderate-to-high in vitro stability, with 39 having
clearance below the lower limit of quantification (LLQ) in
both rat and human hepatocyte assays. 36 showed high in vitro
stability in human hepatocytes but higher clearance in rats,
while 28 showed moderate rat stability. As these values offered
the possibility of promising in vivo pharmacokinetics, all three
compounds were profiled further in vivo in rats.
As predicted from in vitro measurements, the three
compounds all had low measured Clb values. Although the
values are similar, the rank order follows the same pattern as
the in vitro clearance in that 39 had the lowest measured
clearance, followed by 28 with 36 having the highest recorded
value of the three compounds. A more appropriate comparison
of unbound clearance reflects the in vitro data more starkly as
39 has a much higher fraction unbound than the other two
compounds and therefore a significantly lower unbound
clearance.
All three compounds show moderate oral bioavailability in
the rat: 28 has the highest value of the three at 59%. The
reason for the reduced oral bioavailability of 39 and 36 is
unclear, given they have low clearance and good solubility and
permeability but was not investigated further given that these
pharmacokinetic data were sufficient to recommend these tools
for in vivo use.
CONCLUSIONS
■
In conclusion, we were able to replace the embedded aniline
AcK mimetic of 3 (GSK046) with a 1,2,3-triazole motif by
altering the attachment point of the key amide substituent
from the para- to meta-position. This motif was incorporated
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a
Scheme 2. Alternative Routes for the Preparation of Pyridyl Analogues
a
Reagents and conditions: (i) TMS acetylene, Et3N, Cu(I)I (4 mol %), Pd(PPh3)4 (4 mol %), PPh3 (15 mol %), toluene, 80 °C, 3 h (37%); (ii)
NaN3, MeI, pyridine, K2CO3, vitamin C (40 mol %), Cu(II)SO4·5H2O (20 mol %), H2O/MeOH/THF (1:1:1), rt, 18−48 h (19−89%); (iii) TMS
acetylene, iPr2NH, Cu(I)I (4 mol %), Pd(PPh3)4 (2 mol %), THF, rt, 90 min (32%); (iv) (R = Me) LiOH, H2O/THF (1:1), 50 °C, 1 h (98%);
t
(v) (R = Bu) TFA, CHCl3, rt, 6−22 h (42−95%); (vi) R3NH2, HATU or EDC, DIPEA, DMF, rt, 1−16 h; (vii) (X = Br) R2ZnBr or R2ZnCl,
Pd(PPh3)4 (5 mol %), THF, rt, 6.5 h; (viii) (X = Cl) R2ZnBr or R2ZnCl, (PPh3)2Pd(II)Cl2 (10 mol %), THF, 100−110 °C, 30 min to 2 h; (ix)
TFA, CHCl3, rt, 5−21 h (34−66%).
365 nm) or alkaline KMnO4 solution, followed by gentle heating.
LC−MS analysis was carried out on a Waters Acquity UPLC
instrument equipped with a CSH C18 column (50 mm × 2.1 mm, 1.7
μm packing diameter) and Waters micromass ZQ MS using alternate-
scan positive and negative electrospray. Analytes were detected as a
summed UV wavelength of 210−350 nm. Three liquid-phase
methods were used: formic40 °C, 1 mL/min flow rate. Gradient
elution with the mobile phases as (A) H2O containing 0.1% volume/
volume (v/v) formic acid and (B) acetonitrile containing 0.1% (v/v)
formic acid. High pH40 °C, 1 mL/min flow rate. Gradient elution
with the mobile phases as (A) 10 mM aqueous ammonium
bicarbonate solution, adjusted to pH 10 with 0.88 M aqueous
ammonia and (B) acetonitrile. TFA40 °C, 1 mL/min flow rate.
Gradient elution with the mobile phases as (A) 0.1% v/v aqueous
TFA solution and (B) 0.1% v/v TFA solution in acetonitrile. Flash
column chromatography was carried out using Biotage SP4 or Isolera
One apparatus with SNAP silica cartridges. Mass-directed automatic
purification (MDAP) was carried out using a Waters ZQ MS using
alternate-scan positive and negative electrospray and a summed UV
wavelength of 210−350 nm. Two liquid-phase methods were used:
formicSunfire C18 column (100 mm × 19 mm, 5 μm packing
diameter, 20 mL/min flow rate) or Sunfire C18 column (150 mm ×
30 mm, 5 μm packing diameter, 40 mL/min flow rate). Gradient
elution at ambient temperature with the mobile phases as (A) H2O
containing 0.1% volume/volume (v/v) formic acid and (B)
acetonitrile containing 0.1% (v/v) formic acid. High pHXbridge
with both an ortho-benzyloxy and meta-benzyl WPF shelf
group. SAR exploration in the two templates delivered key
analogues with comparable potencies and selectivities to
previously reported series and provided an insight into the
ability to gain further potency (or not) from the ZA channel
using a methyl group. This work culminated with the
identification of three compounds 28 (GSK452), 39
(GSK737), and 36 (GSK217), which had desirable cell
potencies, solubilities, and rat pharmacokinetics. Overall, this
work highlights selective pan-BD2 inhibitors that are
structurally differentiated from the other literature tool
molecules. These inhibitors also provide further structural
insights into strategies to obtain second bromodomain
selectivity.
EXPERIMENTAL SECTION
■
General Experiment. Unless otherwise stated, all reactions were
carried out under an atmosphere of nitrogen in heat- or oven-dried
glassware and anhydrous solvent. Solvents and reagents were
purchased from commercial suppliers and used as received. Reactions
were monitored by thin layer chromatography (TLC) or liquid
chromatography−mass spectrometry (LC−MS). TLC was carried out
on glass or aluminum-backed 60 silica plates coated with a UV254
fluorescent indicator. Spots were visualized using UV light (254 or
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a
Scheme 3. Route for the Preparation of the Pyridyl Indole Compound 36
a
Reagents and conditions: (i) TMS acetylene, Et3N, Cu(I)I (10 mol %), Pd(PPh3)4 (5 mol %), 90 °C, 3 h (72%); (ii) NaN3, MeI, pyridine,
K2CO3, vitamin C (50 mol %), Cu(II)SO4 (20 mol %), H2O/MeOH (1:1), rt, 18−48 h (80%); (iii) mCPBA, CHCl3, rt, 18 h (98%); (iv) TFAA,
50 °C, 12 h (86%); (v) SOCl2, 90 °C, 25 min (65%); (vi) (1H-indol-4-yl)boronic acid, K2CO3, XPhos Pd G1 (5 mol %), XPhos (8 mol %),
toluene/EtOH (9:1), 90 °C, 23 h (10%); (vii) LiOH, H2O/THF (1:1), 50 °C, 90 min (57%); (viii) EtNH2, HATU, DIPEA, DMF, rt, 17 h (39%).
C18 column (100 mm × 19 mm, 5 μm packing diameter, 20 mL/min
flow rate) or Xbridge C18 column (150 mm × 30 mm, 5 μm packing
diameter, 40 mL/min flow rate). Gradient elution at ambient
temperature with the mobile phases as (A) 10 mM aqueous
ammonium bicarbonate solution, adjusted to pH 10 with 0.88 M
aqueous ammonia and (B) acetonitrile. NMR spectra were recorded
at ambient temperature (unless otherwise stated) using standard pulse
methods on any of the following spectrometers and signal
frequencies: Bruker AV-400 (1H = 400 MHz, 13C = 101 MHz),
Bruker AV-600 (1H = 600 MHz, 13C = 150 MHz) or Bruker AV4 700
MHz spectrometer (1H = 700 MHz, 13C = 176 MHz). Chemical
shifts are referenced to TMS or the residual solvent peak and are
reported in ppm. Coupling constants are quoted to the nearest 0.1 Hz
and multiplicities are given by the following abbreviations and
combinations thereof: s (singlet), δ (doublet), t (triplet), q (quartet),
quin (quintet), sxt (sextet), m (multiplet), and br (broad). Liquid
chromatography high-resolution mass spectra (HRMS) were recorded
on a Waters XEVO G2-XS Q-Tof mass spectrometer with the positive
electrospray ionisation mode over a scan rate of 100−200 amu, with
analytes separated on an Acquity UPLC CSH C18 column (100 mm
× 2.1 mm, 1.7 μm packing diameter) at 50 °C. The purity of the
synthesized compounds was determined by LC−MS analysis. All
compounds for biological testing were >95% pure.
Synthetic Procedures. 1-(Benzyloxy)-2-bromobenzene (88). 2-
Bromophenol (0.123 mL, 1.156 mmol), benzyl bromide (0.151 mL,
1.272 mmol), and potassium carbonate (320 mg, 2.312 mmol) were
added to acetone (10 mL), and the reaction mixture was heated at 50
°C for 2 days. The dry residue was dissolved into water and extracted
into EtOAc, which was washed with brine. Brine (20 mL) was added
to the remaining aqueous layer and the compound was extracted into
EtOAc (30 mL). The combined organic layers were dried using a
hydrophobic frit and concentrated in vacuo. The crude product was
then purified by silica chromatography, eluting with 0−15% EtOAc/
cyclohexane. The pure fractions were evaporated in vacuo to give 1-
Figure 5. BromoScan profile of 36 at 10 μM concentration.
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(benzyloxy)-2-bromobenzene (88, 210 mg, 0.798 mmol, 69.0% yield)
mixture was lost. The residue was dissolved in 0.9 mL of dimethyl
sulfoxide (DMSO)/MeOH 1:1 and purified by formic MDAP, and
the relevant fractions were concentrated in vacuo to give 4-(2-
(benzyloxy)phenyl)-1-methyl-1H-1,2,3-triazole (7, 1 mg, 3.77 μmol,
1% yield) as a pale yellow solid. LC−MS (formic, ES+) tR = 1.09 min;
1
as a clear oil. LC−MS (formic, ES+) tR = 1.37 min; m/z = nd; H
NMR (400 MHz, CDCl3-d): δ ppm 7.59 (dd, J = 7.8, 1.5 Hz, 1H)
7.50 (m, 2H) 7.38−7.45 (m, 2H) 7.31−7.38 (m, 1H) 7.25 (td, J =
8.3, 1.0 Hz, 1H) 6.96 (dd, J = 8.3, 1.5 Hz, 1H) 6.87 (td, J = 7.6, 1.5
Hz, 1H) 5.19 (br s, 2H) 1.56 (br s, 1H) 1.45 (s, 1H).
1
m/z = 266.1; (97% pure) H NMR (400 MHz, MeOD-d4): δ ppm
5-(2-(Benzyloxy)phenyl)-3-methylisoxazole (6). 3-Methyl-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (230 mg,
1.100 mmol), 1-(benzyloxy)-2-bromobenzene (88, 210 mg, 0.798
mmol), PdCl2(dppf)-CH2Cl2 adduct (80 mg, 0.098 mmol), and
sodium carbonate (254 mg, 2.394 mmol) were dissolved in 1,4-
dioxane (10 mL) and water (1 mL). The resulting mixture was stirred
at 70 °C for 1.5 h. A further 3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)isoxazole (60 mg, 0.287 mmol), sodium carbonate
(169 mg, 1.596 mmol), and PdCl2(dppf)-CH2Cl2 adduct (80 mg,
0.098 mmol) were added, and the reaction mixture was heated to 170
°C overnight. The reaction was diluted with water and extracted with
EtOAc, including a brine wash. The combined organic fractions were
concentrated in vacuo giving a golden gum. The product was purified
by silica chromatography, eluting with 0−20% EtOAc/cyclohexane.
The fractions were evaporated in vacuo giving a yellowish oil, which
was further purified by formic MDAP. The pure fractions were
concentrated in vacuo, redissolved in MeOH, passed through an NH2
ion-exchange column, and concentrated in vacuo to give 5-(2-
(benzyloxy)phenyl)-3-methylisoxazole (6, 17 mg, 0.064 mmol, 8%
yield) as a white solid. LC−MS (formic, ES+) tR = 1.32 min; m/z =
8.15 (br s, 1H) 8.11 (dd, J = 7.8, 1.5 Hz, 1H) 7.47 (d, J = 6.8 Hz, 2H)
7.39−7.44 (m, 2H) 7.35−7.39 (m, 1H) 7.31 (td, J = 7.9, 1.7 Hz, 1H)
7.16 (d, J = 8.3 Hz, 1H) 7.06 (td, J = 7.8, 1.0 Hz, 1H) 5.26 (s, 2H)
4.09 (br s, 3H).
(E)-N′-(1,1-Dichloropropan-2-ylidene)-4-methylbenzenesulfono-
hydrazide (91). 1,1-Dichloropropan-2-one (0.420 mL, 4.33 mmol)
and para-toluenesulfonhydrazide (0.733 g, 3.94 mmol) were stirred in
propionic acid (5 mL) at room temperature for 4 h, after which the
solid was removed by filtration, washed with cyclohexane, and dried in
a vacuum oven to give (E)-N′-(1,1-dichloropropan-2-ylidene)-4-
methylbenzenesulfonohydrazide (91, 900 mg, 3.05 mmol, 77% yield)
as a white solid. LC−MS (formic, ES+) tR = 0.88 min; m/z = 296.1;
1H NMR (400 MHz, DMSO-d6): δ ppm 11.82 (br s, 1H) 9.19 (s,
1H) 7.80 (dt, J = 8.3, 2.0 Hz, 2H) 7.43 (d, J = 7.8 Hz, 2H) 2.39 (br s,
3H) 1.83 (s, 3H).
1-(2-(Benzyloxy)phenyl)-4-methyl-1H-1,2,3-triazole (8). 2-
(Benzyloxy)aniline (50 mg, 0.251 mmol) in ethanol (3 mL) was
cooled to 0 °C and stirred for 10 min, after which (E)-N′-(1,1-
dichloropropan-2-ylidene)-4-methylbenzenesulfonohydrazide (91, 96
mg, 0.326 mmol) in acetonitrile (2 mL) was added dropwise, and the
reaction mixture was allowed to warm to room temperature and
stirred for 16 h. The reaction mixture was concentrated in vacuo,
taken up into 0.9 mL of 1:1 DMSO/MeOH, and purified by formic
MDAP. The relevant fractions were combined and concentrated in
vacuo to give 1-(2-(benzyloxy)phenyl)-4-methyl-1H-1,2,3-triazole (8,
43 mg, 0.162 mmol, 65% yield) as an orange oil. LC−MS (formic,
ES+) tR = 1.09 min; m/z = 266.2; (100% pure) 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.03 (br s, 1H) 7.63 (dd, J = 7.8, 1.5 Hz, 1H) 7.47
(td, J = 7.3, 2.0 Hz, 1H) 7.27−7.37 (m, 6H) 7.14 (td, J = 7.7, 1.2 Hz,
1H) 5.20 (s, 2H) 2.37 (s, 3H).
1
266.0; (100% pure) H NMR (400 MHz, MeOD-d4): δ ppm 7.90
(dd, J = 7.8, 1.5 Hz, 1H) 7.48−7.53 (m, 2H) 7.34−7.46 (m, 4H)
7.21−7.26 (m, 1H) 7.10 (td, J = 7.6, 1.0 Hz, 1H) 6.65 (s, 1H) 5.28 (s,
3H) 2.25−2.31 (m, 3H).
((2-(Benzyloxy)phenyl)ethynyl)trimethylsilane (89). 1-(Benzyl-
oxy)-2-bromobenzene (88, 0.717 mL, 3.80 mmol), trimethylsilylace-
tylene (2.133 mL, 15.20 mmol), bis(triphenylphosphine)palladium-
(II) chloride (0.267 g, 0.380 mmol), copper(I) iodide (0.072 g, 0.380
mmol), and triethylamine (10 mL, 71.7 mmol) were stirred at 50 °C
for 16 h. Trimethylsilylacetylene (2.133 mL, 15.20 mmol) and
bis(triphenylphosphine)palladium(II) chloride (0.267 g, 0.380 mmol)
were added, and the reaction mixture was stirred for 2 h. The reaction
mixture was cooled to room temperature, filtered through a pad of
celite, and concentrated in vacuo. The residue was purified by silica
chromatography, eluting with 0−10% EtOAc/cyclohexane. The
relevant fractions were concentrated in vacuo to give ((2-(benzyloxy)-
phenyl)ethynyl)trimethylsilane (89, 660 mg, 2.353 mmol, 62% yield)
as an orange oil. LC−MS (formic, ES+) tR = 1.56 min; m/z = 281.1;
1H NMR (400 MHz, MeOD-d4): δ ppm 7.54 (d, J = 7.3 Hz, 2H)
7.28−7.42 (m, 5H) 7.06 (d, J = 7.8 Hz, 1H) 6.93 (td, J = 7.3, 1.0 Hz,
1H) 5.17 (s, 2H) 0.23 (br s, 9H).
Methyl 2-(Benzyloxy)benzoate (92). (Bromomethyl)benzene
(1.093 mL, 9.20 mmol), methyl 2-hydroxybenzoate (0.855 mL,
6.57 mmol), and potassium carbonate (1.272 g, 9.20 mmol) were
stirred in acetone (20 mL) at 50 °C for 24 h. The reaction mixture
was concentrated in vacuo and partitioned between water and EtOAc;
the organic phase was washed with brine, dried using a hydrophobic
frit, and concentrated in vacuo to give methyl 2-(benzyloxy)benzoate
(92, 1.641 g, 6.77 mmol, 76% yield) as a white solid. LC−MS
1
(formic, ES+) tR = 1.18 min; m/z = 243.2; H NMR (400 MHz,
DMSO-d6): δ ppm 7.69 (dd, J = 7.6, 1.7 Hz, 1H) 7.30−7.54 (m, 6H)
7.23 (d, J = 8.8 Hz, 1H) 7.04 (td, J = 6.8, 1.0 Hz, 1H) 5.21 (br s, 2H)
3.81 (s, 3H).
1-(Benzyloxy)-2-ethynylbenzene (90). ((2-(Benzyloxy)phenyl)-
ethynyl)trimethylsilane (89, 514 mg, 1.833 mmol) and potassium
carbonate (380 mg, 2.75 mmol) were stirred in methanol (10 mL) for
1.5 h. The reaction mixture was concentrated in vacuo, diluted with
water, and extracted into EtOAc. The organic layers were combined,
dried via a hydrophobic frit, and concentrated in vacuo. The
compound was isolated by silica chromatography, eluting with 0−
15% EtOAc/cyclohexane. The pure fractions were evaporated in
vacuo to give 1-(benzyloxy)-2-ethynylbenzene (90, 410 mg, 1.969
mmol, 89% yield). LC−MS (formic, ES+) tR = 1.26 min; m/z = 209.2;
1H NMR (400 MHz, MeOD-d4): δ ppm 7.50 (d, J = 7.3 Hz, 2H) 7.43
(dd, J = 7.6, 1.7 Hz, 1H) 7.35−7.41 (m, 2H) 7.31 (td, J = 7.3, 2.4 Hz,
2H) 7.05 (d, J = 8.3 Hz, 1H) 6.93 (td, J = 6.8, 2.4 Hz, 1H) 5.18 (br s,
2H) 3.61 (s, 1H).
2-(Benzyloxy)benzoic Acid (93). Methyl 2-(benzyloxy)benzoate
(92, 1.6 g, 6.60 mmol) and lithium hydroxide (0.237 g, 9.91 mmol)
were stirred in tetrahydrofuran (THF) (10 mL)/water (10 mL) at 50
°C for 16 h and then at 18 °C for 5 h. The reaction mixture was
concentrated in vacuo to remove the THF and was then acidified to
pH 1 with 2 N HCl (aq), a fine precipitate formed, this was extracted
into EtOAc; the organic layer was washed with brine, dried using a
hydrophobic frit, and concentrated in vacuo to give 2-(benzyloxy)-
benzoic acid (93, 1.297 g, 5.68 mmol, 86% yield) as a cream viscous
1
oil. LC−MS (formic, ES+) tR = 1.00 min; m/z 229.1; H NMR (400
MHz, DMSO-d6): δ ppm 12.63 (br s, 1H) 7.67 (dd, J = 7.6, 1.7 Hz,
1H) 7.30−7.54 (m, 6H) 7.19 (d, J = 8.3 Hz, 1H) 7.02 (td, J = 7.3, 1.0
Hz, 1H) 5.20 (s, 2H).
N′-Acetyl-2-(benzyloxy)benzohydrazide (94). 2-(Benzyloxy)-
benzoic acid (93, 370 mg, 1.621 mmol), HATU (678 mg, 1.783
mmol), and DIPEA (0.849 mL, 4.86 mmol) were stirred in
dimethylformamide (DMF) (10 mL) at rt for 5 min, acetohydrazide
(160 mg, 1.945 mmol) was added, and the reaction mixture was
stirred at rt for 16 h. The reaction mixture was diluted with water and
extracted with EtOAc; the organic phase was washed with 10% LiCl
(aq), dried using a hydrophobic frit, and concentrated to give a yellow
oil. This oil was purified by silica chromatography eluting with 20−
4-(2-(Benzyloxy)phenyl)-1-methyl-1H-1,2,3-triazole (7). 1-(Benz-
yloxy)-2-ethynylbenzene (90, 120 mg, 0.576 mmol), sodium azide
(74.9 mg, 1.152 mmol), copper(II) sulfate (18.39 mg, 0.115 mmol),
iodomethane (72.1 μL, 1.152 mmol), and copper powder (36.6 mg,
0.576 mmol) were irradiated at 125 °C in a Biotage microwave at high
absorption for 2 h. Sodium azide (74.9 mg, 1.152 mmol) and
iodomethane (72.1 μL, 1.152 mmol) were added, and the vial was
irradiated at 125 °C in a Biotage microwave at high absorption;
however, due to machine malfunction, the majority of the reaction
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80% EtOAc/cyclohexane. The pure fractions were concentrated in
vacuo to give N′-acetyl-2-(benzyloxy)benzohydrazide (94, 301 mg,
1.059 mmol, 65% yield) as a pale yellow oil. LC−MS (formic, ES+) tR
small portion (78 mg, 0.323 mmol) was purified by formic MDAP.
The appropriate fractions were concentrated under a stream of
nitrogen to give N-(2-(benzyloxy)phenyl)acetamide (11, 59 mg,
0.244 mol, 76% yield) as a white solid. LC−MS (formic, ES+) tR =
0.96 min; m/z = 242; (100% pure) 1H NMR (400 MHz, DMSO-d6):
δ ppm 9.10 (br s, 1H), 7.84 (br d, J = 7.8 Hz, 1H), 7.50 (d, J = 7.3
Hz, 2H), 7.29−7.44 (m, 3H), 6.97−7.11 (m, 2H), 6.89 (t, J = 7.5 Hz,
1H), 5.20 (s, 2H), 2.09 (s, 3H).
Methyl 3-Bromo-4-(methoxymethoxy)benzoate (63). Methyl 3-
bromo-4-hydroxybenzoate (20 g, 87 mmol), MOM-Cl (9.86 mL, 130
mmol), and cesium carbonate (36.7 g, 113 mmol) were stirred in
DMF (200 mL) at rt for 6 h. The reaction mixture was partitioned
between water and EtOAc, and the organic layer was washed with 1 N
NaOH, water, and 10% LiCl (aq), then eluted through a hydrophobic
frit, and concentrated in vacuo to give methyl 3-bromo-4-
(methoxymethoxy)benzoate (63, 23.649 g, 86 mmol, 99% yield) as
a white solid. LC−MS (formic, ES+) tR = 1.12 min; m/z = 276.9; 1H
NMR (400 MHz, DMSO-d6, 295 K): δ ppm 8.11 (d, J = 2.0 Hz, 1H),
7.94 (dd, J = 8.6, 2.2 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 5.40 (s, 2H),
3.84 (s, 3H), 3.42 (s, 3H).
1
= 0.83 min; m/z 285.1; H NMR (400 MHz, DMSO-d6): δ ppm
10.10 (br s, 1H) 9.94 (br s, 1H) 7.69 (dd, J = 7.8, 2.0 Hz, 1H) 7.27−
7.61 (m, 6H) 7.22 (d, J = 7.8 Hz, 1H) 7.01−7.11 (m, 1H) 5.29 (s,
2H) 1.91 (s, 3H).
2-(2-(Benzyloxy)phenyl)-5-methyl-1,3,4-oxadiazole (9). N′-Ace-
tyl-2-(benzyloxy)benzohydrazide (94, 301 mg, 1.059 mmol) and
POCl3 (5 mL, 53.6 mmol) were stirred at 110 °C for 1 h. POCl3 was
removed in vacuo azeotroping with toluene (×2); the resulting yellow
oil was purified using silica chromatography eluting with a gradient of
0−100% EtOAc/cyclohexane. The relevant fractions were concen-
trated in vacuo and the material was further purified using formic
MDAP. The pure fractions were concentrated in vacuo to give 2-(2-
(benzyloxy)phenyl)-5-methyl-1,3,4-oxadiazole (9, 51 mg, 0.192
mmol, 18% yield) as a light green oil. LC−MS (formic, ES+) tR
=
1
1.06 min; m/z = 267.1; (100% pure) H NMR (400 MHz, DMSO-
d6): δ ppm 7.84 (dd, J = 7.6, 1.7 Hz, 1H) 7.49−7.62 (m, 3H) 7.37−
7.45 (m, 2H) 7.30−7.37 (m, 2H) 7.14 (td, J = 7.6, 1.0 Hz, 1H) 5.30
(s, 2H) 2.57 (s, 3H).
Methyl 4-(Methoxymethoxy)-3-((trimethylsilyl)ethynyl)benzoate
(64). Methyl 3-bromo-4-(methoxymethoxy)benzoate (63, 23 g, 84
mmol), ethynyltrimethylsilane (60 mL, 425 mmol), Et3N (100 mL,
717 mmol), PdCl2(PPh3)2 (2.93 g, 4.18 mmol), and copper iodide
(1.592 g, 8.36 mmol) were combined under nitrogen, and the mixture
was heated at 60 °C for 24 h. The reaction mixture was diluted with
EtOAc (200 mL) and filtered, and the filtrate was then washed with
water (2 × 200 mL), dried with sodium sulfate, and concentrated in
vacuo. The resulting dark brown oil was purified by silica
chromatography eluting with 0−25% EtOAc/cyclohexane. The
product-containing fractions were concentrated in vacuo to give
methyl 4-(methoxymethoxy)-3-((trimethylsilyl)ethynyl)benzoate
(64, 22.3 g, 76 mmol, 91% yield) as a brown oil. LC−MS (formic,
2-(5-Methyl-1H-1,2,4-triazol-3-yl)phenol (95). A mixture of 2-
(benzyloxy) benzonitrile (0.5 g, 2.38 mmol), acetohydrazide (176.3
mg, 2.38 mmol), and potassium carbonate (98 mg, 0.714 mmol) in
nBuOH (4 mL) was heated to 150 °C for 6 h under microwave
conditions. The reaction mixture was concentrated under reduced
pressure; crude residue was diluted with water (20 mL) and extracted
with EtOAc (2 × 30 mL). The organic layer was dried over anhydrous
Na2SO4, filtered, concentrated, and dried to get 500 mg of the crude
compound. The crude compound was purified by silica chromatog-
raphy eluting with 0−30% EtOAc in pet ether. The pure fractions
were collected, concentrated, and dried to give 2-(5-methyl-1H-1,2,4-
triazol-3-yl)phenol (95, 120 mg, 0.616 mmol, 26% yield) as an off-
white solid. LC−MS (high pH, ES+) tR = 3.38 min; m/z = 176.0; 1H
NMR (400 MHz, CDCl3-d): δ ppm 11.01 (br s, 1H) 7.88 (br d, J =
7.0 Hz, 1H) 7.32 (td, J = 7.0, 1.5 Hz, 1H) 7.04 (d, J = 7.7 Hz, 1H)
6.92 (td, J = 7.2, 1.0 Hz, 1H) 2.55 (s, 3H).
1
ES+) tR = 1.41 min; m/z = 293; H NMR (400 MHz, CDCl3-d, 295
K): δ ppm 8.15 (d, J = 2.4 Hz, 1H), 7.93−7.98 (dd, J = 8.8, 2.4 Hz,
1H), 7.13 (d, J = 8.8 Hz, 1H), 5.31 (s, 2H), 3.91 (s, 3H), 3.51−3.56
(m, 3H), 0.24−0.32 (m, 9H).
3-(2-(Benzyloxy)phenyl)-5-methyl-1H-1,2,4-triazole (10). 2-(5-
Methyl-1H-1,2,4-triazol-3-yl)phenol (95, 70 mg, 0.400 mmol),
potassium carbonate (66.3 mg, 0.479 mmol), and benzyl bromide
(0.048 mL, 0.400 mmol) were dissolved in acetone (8 mL) at rt, and
the reaction mixture was stirred at 20 °C for 4 h. Then, the reaction
mixture was heated to 40 °C for 4 h. The reaction mixture was
quenched with ice-cold water (20 mL) and extracted with EtOAc (2
× 30 mL). The organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated to get 70 mg of the crude compound. This
crude compound was purified by combining with the crude
compound to get 120 mg of the crude compound. The crude
compound was purified by silica chromatography eluting with 0−30%
EtOAc in pet ether. The relevant fractions were collected,
concentrated, and dried to get an impure material. This was further
purified by formic MDAP. The pure fraction was lyophilized and
dried to get 3-(2-(benzyloxy)phenyl)-5-methyl-1H-1,2,4-triazole (10,
10 mg, 0.038 mmol, 10% yield) as a white solid. LC−MS (formic,
ES+) tR = 4.30 min; m/z = 266.3; (100% pure) 1H NMR (400 MHz,
CDCl3-d): δ ppm 11.31 (br s, 1H) 8.33 (dd, J = 8.1, 1.5 Hz, 1H)
7.37−7.51 (m, 6H) 7.09−7.18 (m, 2H) 5.24 (s, 2H) 2.45 (s, 3H).
N-(2-(Benzyloxy)phenyl)acetamide (11). A mixture of acetamido-
phenol (154.17 g, 1.0 mol), potassium carbonate (138.2 g, 1.00 mol),
sodium iodide (14.9 g, 0.1 mol), and benzyl bromide (125 mL, 1.05
mol) was combined in acetone (1 L) under nitrogen and heated to
reflux for 4 h. The solvent was reduced to half volume by distillation
at atmospheric pressure, and the cooled reaction mixture was
partitioned between EtOAc (3 L) and water (500 mL). The organic
layer was washed with 1:1 brine: water (500 mL) and then the solvent
was removed by distillation at atmospheric pressure to give a brown
solid. The solid was triturated with cyclohexane (450 mL) and
collected by filtration. The filter cake was washed with cyclohexane (3
× 100 mL) and then dried under vacuum at room temperature to give
N-(2-(benzyloxy)phenyl)acetamide (216.2 g, 0.9 mol, 89.6% yield). A
General Procedure A. Methyl 4-(Methoxymethoxy)-3-(1-meth-
yl-1H-1,2,3-triazol-4-yl)benzoate (57). Sodium azide (6.67 g, 103
mmol), ascorbic acid (4.82 g, 27.4 mmol), potassium carbonate
(17.02 g, 123 mmol), and copper(II) sulfate (2.183 g, 13.68 mmol)
were combined and then water (160 mL) and methanol (160 mL)
were added. The mixture was stirred at room temperature. Methyl 4-
(methoxymethoxy)-3-((trimethylsilyl)ethynyl)benzoate (64, 20 g,
68.4 mmol), MeI (12.83 mL, 205 mmol), and pyridine (27.7 mL,
342 mmol) were added, and the mixture was stirred at room
temperature over 60 h. The mixture was evaporated to approximately
half volume and then extracted with dichloromethane (DCM) (3 ×
200 mL), and the combined organics were washed with brine, then
dried, and evaporated in vacuo to give a dark brown oil. The crude
product was dissolved in DCM and purified by silica chromatography
eluting with 0−100% EtOAc/cyclohexane, and the product-
containing fractions were evaporated in vacuo to give methyl 4-
(methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoate (57,
15 g, 54.1 mmol, 79% yield). LC−MS (formic, ES+) tR = 0.86 min;
m/z = 278.2; 1H NMR (400 MHz, CDCl3-d): δ ppm 9.03 (d, J = 2.0
Hz, 1H) 7.98−8.04 (m, 2H) 7.24 (d, J = 8.8 Hz, 1H) 5.39 (s, 2H)
4.19 (s, 3H) 3.94 (s, 3H) 3.52 (s, 3H).
4-(Methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic
Acid (96). A suspension of methyl 4-(methoxymethoxy)-3-(1-methyl-
1H-1,2,3-triazol-4-yl)benzoate (57, 11 g, 39.7 mmol) in THF (40
mL), water (40 mL), and NaOH (39.7 mL, 79 mmol) was stirred at rt
for 4 h. The reaction mixture was concentrated and taken up into
water. Acetic acid was added until pH 5, precipitating a gray solid,
which was isolated by vacuum filtration through a sinter funnel. The
filter cake was washed with water and dried in a vacuum oven to give
4-(methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic acid
(96, 8.26 g, 31.4 mmol, 79% yield). LC−MS (formic, ES+) tR
0.69 min; m/z = 264.1.
=
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4-Hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic Acid (97).
4-(Methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic acid
(96, 2 g, 7.60 mmol) and conc HCl (1 mL, 32.9 mmol) were stirred
at 50 °C for 16 h. A precipitate had formed in the reaction, this was
removed by filtration and dried to give 4-hydroxy-3-(1-methyl-1H-
1,2,3-triazol-4-yl)benzoic acid (97, 1.432 g, 6.53 mmol, 86% yield) as
a cream solid. LC−MS (formic, ES+) tR = 0.65 min; m/z = 220.1; 1H
NMR (400 MHz, DMSO-d6): δ ppm 12.53 (br s, 1H) 10.97 (s, 1H)
8.66 (d, J = 2.4 Hz, 1H) 8.43 (s, 1H) 7.72−7.79 (m, 1H) 7.03 (d, J =
8.8 Hz, 1H) 4.11 (s, 3H).
Methyl 4-(Benzyloxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoate
(98). 4-Hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic acid (97,
2.498 g, 11.40 mmol), benzyl bromide (2.710 mL, 22.79 mmol), and
potassium carbonate (4.730 g, 34.20 mmol) were stirred at rt in
acetone (20 mL) for 16 h. The reaction mixture was concentrated in
vacuo, and the residue was partitioned between water (25 mL) and
EtOAc (25 mL). The organic layer was washed with brine (25 mL),
dried using a hydrophobic frit, and concentrated to give benzyl 4-
(benzyloxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoate (98, 4.230 g,
10.59 mmol, 93% yield) as a beige solid. LC−MS (formic, ES+) tR =
1.32 min; m/z = 400.
4-(Benzyloxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic Acid
(99). Benzyl 4-(benzyloxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-
benzoate (98, 4.23 g, 10.59 mmol) and lithium hydroxide (0.254 g,
10.59 mmol) were combined in THF (20 mL)/water (20 mL) and
stirred at 50 °C for 16 h. The reaction mixture was concentrated in
vacuo to remove the THF and was acidified to pH 2 with 2 N HCl
(aq). The resulting precipitate was collected by filtration and dried to
give 4-(benzyloxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic acid
(99, 3.08 g, 9.96 mmol, 94% yield) as a cream solid. LC−MS
(formic, ES+) tR = 0.94 min; m/z = 310; 1H NMR (400 MHz,
DMSO-d6): δ ppm 12.73 (br s, 1H), 8.75 (d, J = 2.0 Hz, 1H), 8.31 (s,
1H), 7.87 (dd, J = 8.8, 2.4 Hz, 1H), 7.47−7.55 (m, 2H), 7.26−7.47
(m, 4H), 5.42 (s, 2H), 4.09 (s, 3H).
4-(Benzyloxy)-N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-
benzamide (13). 4-(Benzyloxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-
benzoic acid (99, 2.6 g, 8.41 mmol) was taken up into DMF (65
mL). DIPEA (5.87 mL, 33.6 mmol) was added, and the mixture was
left to stir for 10 s. HATU (4.79 g, 12.61 mmol) was then added, and
the mixture was left to stir for 1 min. 2 M ethanamine in THF (5.04
mL, 10.09 mmol) was added, and the reaction mixture was stirred at
room temperature for 2 h. The mixture was concentrated in vacuo to
give a brown oil. The oil was partitioned between EtOAc and water
(30 mL each), and the organic phase was washed with 2 M NaOH
(60 mL), filtered through a hydrophobic frit, and concentrated in
vacuo to give 4-(benzyloxy)-N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-
yl)benzamide (13, 2.80 g, 8.32 mmol, 99% yield) as a pale yellow
solid. LC−MS (formic, ES+) tR = 0.95 min; m/z = 337; (100% pure)
1H NMR (400 MHz, MeOD-d4): δ ppm 7.80 (dd, J = 9.3, 2.4 Hz,
yield) as an orange oil. LC−MS (formic, ES+) tR = 1.63 min; m/z =
1
310; H NMR (400 MHz, CDCl3-d): δ ppm 7.80−7.88 (m, 1H),
7.69−7.80 (m, 1H), 1.45 (s, 9H), 0.29 (s, 9H).
tert-Butyl 2-Chloro-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (69). General procedure A was followed using the
following amounts: methyl iodide (0.282 mL, 4.52 mmol), tert-butyl
2-chloro-6-((trimethylsilyl)ethynyl)isonicotinate (80, 1.166 g, 1.505
mmol), sodium azide (0.215 g, 3.31 mmol), vitamin C (0.106 g, 0.602
mmol), potassium carbonate (0.374 g, 2.71 mmol), pyridine (0.609
mL, 7.53 mmol), and copper sulfate pentahydrate (0.075 g, 0.301
mmol) in methanol (4 mL), THF (4.00 mL), and water (4.00 mL).
This gave tert-butyl 2-chloro-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (69, 211.5 mg, 0.718 mmol, 48% yield) as a cream
1
solid. LC−MS (formic, ES+) tR = 1.19 min; m/z = 295.1; H NMR
(400 MHz, CDCl3-d): δ ppm 8.55 (d, J = 1.0 Hz, 1H) 8.19 (s, 1H)
7.76 (d, J = 1.5 Hz, 1H) 4.19 (s, 3H) 1.64 (s, 9H).
2-Chloro-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic Acid
(100). TFA (0.971 mL, 12.61 mmol) was added to a solution of
tert-butyl 2-chloro-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate
(69, 464.5 mg, 1.576 mmol) in DCM (7 mL), and the reaction
mixture was stirred at room temperature under nitrogen for 3 h after
which further TFA (0.971 mL, 12.61 mmol) was added. Stirring
continued for 1 h after which further TFA (0.971 mL, 12.61 mmol)
was added, and the reaction mixture was stirred for 2 h and left to
stand at room temperature for 16 h. The reaction mixture was
concentrated and the product was left to dry in vacuo at 40 °C for 3 h
to give 2-chloro-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic acid
(100, 437 mg, 1.557 mmol, 99% yield) as a brown solid. LC−MS
(formic, ES+) tR = 0.76 min; m/z = 239; 1H NMR (400 MHz,
DMSO-d6, 303 K): δ ppm 8.72 (s, 1H), 8.37 (d, J = 1.5 Hz, 1H), 7.77
(d, J = 1.0 Hz, 1H), 4.13 (s, 3H) (carboxylic acid not visible in
spectrum).
2-Chloro-N-ethyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (71). 2 M ethanamine in THF (0.758 mL, 1.516
mmol) was added to a solution of 2-chloro-6-(1-methyl-1H-1,2,3-
triazol-4-yl)isonicotinic acid (100, 428 mg, 1.166 mmol), HATU
(576 mg, 1.516 mmol), and DIPEA (0.407 mL, 2.332 mmol) in DMF
(7 mL). The reaction mixture was stirred at room temperature under
nitrogen for 2 h and left to stand in solution for 16 h. The reaction
mixture was partitioned between ethyl acetate (30 mL) and water (20
mL), the organic layer was washed with water (10 mL) and 10% LiCl
solution (10 mL) and then passed through a hydrophobic frit, and the
solvent was removed in vacuo. The resulting oil was dissolved in DCM
and purified by silica chromatography eluting with 0−70% ethyl
acetate/cyclohexane. The product-containing fractions were com-
bined, and the solvent was removed in vacuo to give 2-chloro-N-ethyl-
6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinamide (71, 251.5 mg,
0.852 mmol, 73% yield) as a pale yellow solid. LC−MS (formic,
1
ES+) tR = 0.73 min; m/z = 266; H NMR (400 MHz, CDCl3-d): δ
1H), 7.34−7.51 (m, 5H), 7.23 (d, J = 8.8 Hz, 1H), 5.34 (s, 2H),
4.82−4.82 (m, 5H), 4.10 (s, 3H), 3.43 (q, J = 7.3 Hz, 2H), 3.34−3.38
(m, 1H), 2.94−3.09 (m, 1H), 1.25 (t, J = 6.4 Hz, 3H).
ppm 8.27 (d, J = 1.5 Hz, 1H), 8.21 (s, 1H), 7.73 (d, J = 1.5 Hz, 1H),
6.44 (br s, 1H), 4.20 (s, 3H), 3.48−3.62 (m, 3H), 1.30 (t, J = 6.9 Hz,
3H).
tert-Butyl 2-Chloro-6-((trimethylsilyl)ethynyl)isonicotinate (80).
tert-Butyl 2,6-dichloroisonicotinate (2 g, 8.06 mmol), Pd(PPh3)4
(0.373 g, 0.322 mmol), triethylamine (5.2 mL, 37.3 mmol),
triphenylphosphine (0.317 g, 1.209 mmol), and copper iodide
(0.061 g, 0.322 mmol) were combined in toluene (13 mL) in a
microwave vial, and the vial was vacuum-degassed and flushed with
nitrogen. Ethynyltrimethylsilane (1.139 mL, 8.06 mmol) was added,
and the vial was again degassed and flushed with nitrogen. The
reaction mixture was heated to 80 °C for 6.5 h under microwave
conditions and then left to stand in solution overnight. The reaction
mixture was partitioned between ethyl acetate (100 mL) and water
(40 mL). The organic layer was washed with water (40 mL) and brine
(40 mL), then passed through a hydrophobic frit, and concentrated in
vacuo. The resulting oil was suspended in cyclohexane and purified by
silica gel chromatography using a gradient of 0−20% ethyl acetate/
cyclohexane. The product-containing fractions were combined, and
the solvent was removed in vacuo to give tert-butyl 2-chloro-6-
((trimethylsilyl)ethynyl)isonicotinate (80, 6.574 g, 7.00 mmol, 87%
2-Benzyl-N-ethyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (14). PdCl2(PPh3)2 (52.8 mg, 0.075 mmol) and 2-
chloro-N-ethyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinamide
(71, 100 mg, 0.376 mmol) were dissolved in THF (3 mL) in a
microwave vial which was then sealed, and benzylzinc(II) bromide 0.5
M in THF (1.505 mL, 0.753 mmol) was added. The reaction mixture
was heated to 110 °C for 1 h under microwave conditions. The
reaction mixture was partitioned between ethyl acetate (30 mL) and
water (10 mL), and the organic layer was washed with water (10 mL)
and brine (10 mL). The organic layer was passed through a
hydrophobic frit, and the solvent was removed in vacuo. The resulting
solid was dissolved in DCM and purified by silica chromatography
eluting with 0−80% ethyl acetate/cyclohexane followed by 0−4%
ethyl acetate/ethanol. The product-containing fractions were
combined, and the solvent was removed in vacuo to give a pale
brown solid. The product was further purified by formic MDAP and
the product-containing fraction was concentrated under a stream of
nitrogen to give 2-benzyl-N-ethyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
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isonicotinamide (14, 6.6 mg, 0.021 mmol, 5.46% yield) as a white
solid. LC−MS (formic, ES+) tR = 0.91 min; m/z = 322; (100% pure)
1H NMR (400 MHz, CDCl3-d): δ ppm 8.18 (s, 1H). 8.17 (d, J = 1.5
Hz, 1H), 7.56 (d, J = 1.5 Hz, 1H), 7.29−7.35 (m, 4H), 7.21−7.27 (m,
1H), 6.30 (br s, 1H), 4.23 (2H, s), 4.20 (s, 3H), 3.52 (qd, J = 7.3, 5.6
Hz, 3H), 1.28 (t, J = 7.3 Hz, 4H).
mL) and water (75 mL). The organic layer was washed with water
(50 mL) and brine (50 mL) and passed through a hydrophobic frit,
and the solvent was removed in vacuo. The resulting oily solid was
dissolved in DCM and loaded onto a 340 g Biotage SNAP silica
column which was left to dry overnight. The product was eluted with
a gradient of 0−20% cyclohexane/EtOAc, the product-containing
fractions were combined, and the solvent was removed in vacuo. The
product was left to dry in vacuo for 1 h to give methyl 2-bromo-6-
((trimethylsilyl)ethynyl)isonicotinate (81, 3.5466 g, 5.68 mmol, 34%
yield) as an orange oil. LC−MS (formic, ES+) tR = 1.47 min; m/z =
Methyl 3-Ethynyl-4-(methoxymethoxy)benzoate (101). Methyl
4-(methoxymethoxy)-3-((trimethylsilyl)ethynyl)benzoate (64, 2.61 g,
8.93 mmol) and potassium carbonate (2.96 g, 21.42 mmol) were
stirred in methanol (20 mL) for 40 min. The reaction mixture was
diluted with 2 M HCl and extracted into DCM, including a brine
wash. The organic layers were then combined, dried via a
hydrophobic frit, and concentrated in vacuo. The residue was taken
up into DCM and purified by silica chromatography eluting with 20−
70% EtOAc/cyclohexane, and the relevant fractions were concen-
trated to give methyl 3-ethynyl-4-(methoxymethoxy)benzoate (101,
600 mg, 2.72 mmol, 31% yield). LC−MS (formic, ES+) tR = 1.01 min;
m/z = 221.1.
N-Ethyl-4-(methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-
benzamide (58). 4-(Methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-
4-yl)benzoic acid (96, 1.60 g, 6.08 mmol), HATU (2.54 g, 6.69
mmol) and DIPEA (2.123 mL, 12.16 mmol) were stirred in DMF (10
mL) at rt for 10 min, ethanamine (4.56 mL, 9.12 mmol) was added,
and the reaction mixture was stirred at rt for 16 h. The reaction
mixture was partitioned between water and EtOAc, the organic layer
was washed with 10% LiCl (aq), dried using a hydrophobic frit, and
concentrated to a cream solid. This solid was purified by silica
chromatography eluting with 0−5% 2 M NH3 in MeOH/DCM to
give N-ethyl-4-(methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-
benzamide (58, 1.160 g, 4.00 mmol, 66% yield) as a cream solid. LC−
MS (formic, ES+) tR = 0.69 min; m/z = 291.1; (98% pure).
N-Ethyl-4-hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzamide
(102). N-Ethyl-4-(methoxymethoxy)-3-(1-methyl-1H-1,2,3-triazol-4-
yl)benzamide (58, 85 mg, 0.293 mmol) and hydrochloric acid (1
mL, 12.00 mmol) were stirred in methanol (10 mL) at rt for 1 h. The
reaction was warmed to 50 °C and stirred for 1.5 h. The reaction
mixture was neutralized with NaOH and concentrated in vacuo. The
residue was taken up into water and extracted into DCM, including a
brine wash. The organic extracts were combined, dried via a
hydrophobic frit, and concentrated in vacuo, giving N-ethyl-4-
hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzamide (102, 71 mg,
0.288 mmol, 98% yield) as a white solid. LC−MS (formic, ES+) tR =
0.64 min; m/z = 247.1.
1
314.0; H NMR (400 MHz, CDCl3-d): δ 7.94 (d, J = 1.5 Hz, 1H)
7.91 (d, J = 1.5 Hz, 1H) 3.95 (s, 3H) 0.26 (s, 9H).
Methyl 2-Bromo-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate
(70). General procedure A was followed using the following amounts:
pyridine (2.297 mL, 28.4 mmol), methyl 2-bromo-6-((trimethylsilyl)-
ethynyl)isonicotinate (81, 3.5466 g, 5.68 mmol), sodium azide (0.812
g, 12.49 mmol), vitamin C (0.400 g, 2.272 mmol), methyl iodide
(1.065 mL, 17.04 mmol), potassium carbonate (1.413 g, 10.22
mmol), and copper(II) sulphate pentahydrate (0.284 g, 1.136 mmol)
in methanol (6 mL), THF (6 mL), and water (6 mL). This gave
methyl 2-bromo-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate (70,
615 mg, 1.656 mmol, 29% yield) as a pale yellow solid. LC−MS
1
(formic, ES+) tR = 0.93 min; m/z = 299.0; H NMR (400 MHz,
CDCl3-d): δ 8.67 (d, J = 1.5 Hz, 1H) 8.21 (s, 1H) 7.97 (d, J = 1.5 Hz,
1H) 4.20 (s, 3H) 4.00 (s, 3H).
Methyl 2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate
(74). General procedure A was followed using the following amounts:
iodomethane (1.015 mL, 16.23 mmol), potassium carbonate (1.346 g,
9.74 mmol), sodium azide (0.774 g, 11.90 mmol), methyl 2-benzyl-6-
((trimethylsilyl)ethynyl)isonicotinate (132, 1.75 g, 5.41 mmol), L-
ascorbic acid (0.381 g, 2.164 mmol), copper(II) sulfate pentahydrate
(0.270 g, 1.082 mmol), and pyridine (2.188 mL, 27.1 mmol) in water
(20 mL), methanol (20 mL), and THF (10 mL). This gave methyl 2-
benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate (74, 958 mg,
2.80 mmol, 52% yield) as a pale brown solid. LC−MS (TFA, ES+) tR
= 1.01 min; m/z = 309.2; 1H NMR (400 MHz, DMSO-d6): δ 8.63 (s,
1H) 8.28 (d, J = 1.5 Hz, 1H) 7.64 (d, J = 1.5 Hz, 1H) 7.37−7.29 (m,
4H) 7.22 (tt, J = 6.8, 1.5 Hz, 1H) 4.23 (s, 2H) 4.13 (s, 3H) 3.91 (s,
3H).
2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic Acid (82).
Lithium hydroxide (19.57 mg, 0.817 mmol) in water (3 mL) was
added to a solution of methyl 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-
4-yl)isonicotinate (74, 420 mg, 0.409 mmol) in THF (3 mL). The
reaction mixture was heated to 50 °C under nitrogen for 1 h. The
reaction mixture was left to cool, passed through a hydrophobic frit,
and THF was removed under reduced pressure. The remaining
solution was acidified to pH 2 with 2 M HCl and a precipitate formed.
The precipitate was collected by filtration and dried in vacuo to give 2-
benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic acid (82, 182
mg, 0.402 mmol, 98% yield) as an orange solid. LC−MS (formic,
N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(pyridin-2-
ylmethoxy)benzamide (15). N-Ethyl-4-hydroxy-3-(1-methyl-1H-
1,2,3-triazol-4-yl)benzamide (102, 71 mg, 0.288 mmol), 2-
(bromomethyl)pyridine, hydrobromide (80 mg, 0.317 mmol), and
potassium carbonate (120 mg, 0.865 mmol) were stirred in acetone
(10 mL) at rt for 67 h. The reaction mixture was concentrated in
vacuo, partitioned between water and DCM, and extracted into DCM,
including a brine wash. The organic extracts were combined, dried via
a hydrophobic frit, and concentrated in vacuo. The residue was taken
up into DCM and purified by silica chromatography eluting 0−5%
MeOH/DCM and the relevant fractions were combined and
concentrated in vacuo to give N-ethyl-3-(1-methyl-1H-1,2,3-triazol-
4-yl)-4-(pyridin-2-ylmethoxy)benzamide (15, 78 mg, 0.231 mmol,
80% yield) as a white solid. LC−MS (formic, ES+) tR = 0.63 min; m/z
1
ES+) tR = 0.91 min; m/z 295.2; H NMR (400 MHz, CDCl3-d): δ
ppm 8.66 (s, 1H) 8.20 (br d, J = 7.8 Hz, 1H) 7.66−7.75 (m, 1H)
7.17−7.35 (m, 5H) 4.23−4.29 (m, 2H) 4.18−4.22 (m, 3H).
2-Benzyl-N-cyclopropyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (16). DIPEA (0.031 mL, 0.177 mmol) was added to
a solution of 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic
acid (82, 40 mg, 0.088 mmol), cyclopropanamine (7.96 μL, 0.115
mmol), and HATU (43.7 mg, 0.115 mmol) in DMF (1 mL). The
reaction mixture was stirred at room temperature under nitrogen for 1
h. The reaction mixture was purified by formic MDAP and the
product-containing fraction was concentrated. The product was left to
dry in vacuo for 2 days to give 2-benzyl-N-cyclopropyl-6-(1-methyl-
1H-1,2,3-triazol-4-yl)isonicotinamide (16, 19.5 mg, 0.058 mmol, 66%
yield) as a pale yellow solid. LC−MS (formic, ES+) tR = 0.92 min; m/
z = 334.2; (100% pure) 1H NMR (400 MHz, CDCl3-d): δ ppm 8.42
(s, 1H) 8.23 (d, J = 1.0 Hz, 1H) 7.74 (s, 1H) 7.28−7.42 (m, 5H) 6.75
(br s, 1H) 4.33 (s, 2H) 4.21 (s, 3H) 2.93−3.01 (m, 1H) 0.86−0.96
(m, 2H) 0.69−0.78 (m, 2H).
1
= 338.2; (100% pure) H NMR (400 MHz, MeOD-d4): δ ppm 8.62
(m, J = 2.4 Hz, 2H) 8.38 (s, 1H) 7.88 (td, J = 7.7, 1.7 Hz, 1H) 7.80
(dd, J = 8.8, 2.4 Hz, 1H) 7.56 (d, J = 7.8 Hz, 1H) 7.38−7.44 (m, 1H)
7.21 (d, J = 8.3 Hz, 1H) 5.44 (s, 2H) 4.15 (s, 3H) 3.43 (q, J = 7.3 Hz,
2H) 1.25 (t, J = 7.3 Hz, 3H).
Methyl 2-Bromo-6-((trimethylsilyl)ethynyl)isonicotinate (81).
Methyl 2,6-dibromoisonicotinate (5 g, 16.95 mmol), copper iodide
(0.129 g, 0.678 mmol), diisopropylamine (14.50 mL, 102 mmol), and
tetrakis (0.392 g, 0.339 mmol) in THF (35 mL) were stirred in a
three-necked flask at rt and vacuum-degassed with N2. Ethynyl-
trimethylsilane (2.156 mL, 15.26 mmol) was then added via syringe
and stirring continued under nitrogen for 1 h. The reaction mixture
was quenched with 2 M HCl and partitioned between EtOAc (75
(S)-Methyl 3-Bromo-4-(1-phenylethoxy)benzoate (103). Tri-N-
butylphosphine (1.410 mL, 5.71 mmol), (R)-1-phenylethanol (0.690
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mL, 5.71 mmol), and methyl 3-bromo-4-hydroxybenzoate (1.1 g, 4.76
mmol) were stirred in toluene (30 mL) under nitrogen. (E)-Diazene-
1,2-diylbis(piperidin-1-ylmethanone) (1.442 g, 5.71 mmol) was
added over 10 min, and the reaction mixture was stirred at rt for
17 h. The solvent was removed in vacuo, and the compound was
dissolved in DCM and purified by silica chromatography eluting with
0−50% EtOAc/cyclohexane. The product-containing fractions were
concentrated in vacuo to give (S)-methyl 3-bromo-4-(1-
phenylethoxy)benzoate (103, 1.8 g, 5.37 mmol, 113% yield). LC−
with 1 M aqueous HCl (1 mL) and extracted into DCM (2 × 5 mL).
The organic extracts were combined, eluted through a hydrophobic
frit, and concentrated in vacuo. The residue was purified by formic
MDAP, and the product-containing fractions were concentrated in
vacuo to give (S)-N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-
phenylethoxy)benzamide (17, 35 mg, 0.100 mmol, 84% yield).
1
LC−MS (formic, ES+) tR = 0.99 min; m/z = 351; (97% pure) H
NMR (400 MHz, MeOD-d4): δ ppm 8.60 (d, J = 2.0 Hz, 1H), 8.38
(s, 1H), 7.61 (dd, J = 8.8, 2.4 Hz, 1H), 7.22−7.39 (m, 6H), 6.96 (d, J
= 8.8 Hz, 1H), 5.64 (q, J = 6.4 Hz, 1H), 4.20 (s, 3H), 3.40 (q, J = 7.3
Hz, 2H), 1.76 (d, J = 6.4 Hz, 3H), 1.22 (t, J = 7.3 Hz, 3H).
N-Ethyl-2-(1-methyl-1H-1,2,3-triazol-4-yl)-6-(1phenylethyl)-
isonicotinamide (18). (1-Phenylethyl)zinc(II) bromide 0.5 M in
THF (0.514 mL, 0.257 mmol) was added to a sealed microwave vial
containing 2-chloro-N-ethyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (71, 70 mg, 0.171 mmol) and PdCl2(PPh3)2 (12.02
mg, 0.017 mmol) in THF (3 mL). The reaction mixture was heated to
110 °C for 60 min under microwave conditions after which further
PdCl2(PPh3)2 (12.02 mg, 0.017 mmol) and (1-phenylethyl)zinc(II)
bromide 0.5 M in THF (0.514 mL, 0.257 mmol) were added. Heating
continued for 35 min under microwave conditions after which further
(1-phenylethyl)zinc(II) bromide 0.5 M in THF (0.514 mL, 0.257
mmol) and PdCl2(PPh3)2 (12.02 mg, 0.017 mmol) were added.
Heating continued for 30 min under microwave conditions. The
reaction mixture was partitioned between ethyl acetate (30 mL) and
water (20 mL), and the organic layer was washed with water (2 × 10
mL) and brine (10 mL). The organic layer was passed through a
hydrophobic frit, and the solvent was removed in vacuo. The resulting
solid was dissolved in DCM and purified by silica chromatography
eluting with 30−100% ethyl acetate/heptane. The impure product-
containing fractions were combined, and the solvent was removed in
vacuo. The impure product was dissolved in 1:1 DMSO/methanol
and purified by formic MDAP. The product-containing fraction was
concentrated and left to dry in vacuo for 4 h to give N-ethyl-2-(1-
methyl-1H-1,2,3-triazol-4-yl)-6-(1-phenylethyl)isonicotinamide (18,
11.7 mg, 0.035 mmol, 20% yield) as a white solid. LC−MS (formic,
ES+) tR = 1.00 min; m/z = 336.2; (100% pure) 1H NMR (400 MHz,
DMSO-d6): δ ppm 8.84 (br t, J = 5.4 Hz, 1H) 8.61 (s, 1H) 8.23 (d, J
= 1.5 Hz, 1H) 7.58 (d, J = 1.5 Hz, 1H) 7.34−7.41 (m, 2H) 7.25−7.34
(m, 2H) 7.19 (tt, J = 6.8, 1.0 Hz, 1H) 4.36 (q, J = 7.0 Hz, 1H) 4.14 (s,
3H) 3.22−3.37 (m, 2H) 1.69 (d, J = 6.8 Hz, 3H) 1.14 (t, J = 7.1 Hz,
3H).
1
MS (formic, ES+) tR = 1.43 min; m/z = not seen; H NMR (400
MHz, CDCl3-d): δ ppm 8.24 (d, J = 2.0 Hz, 1H), 7.81 (dd, J = 8.6,
2.2 Hz, 1H), 7.27−7.42 (m, 5H), 6.77 (d, J = 8.3 Hz, 1H), 5.46 (q, J
= 6.4 Hz, 1H), 3.87 (s, 3H), 1.74 (d, J = 6.4 Hz, 3H).
(S)-Methyl 4-(1-Phenylethoxy)-3-((trimethylsilyl)ethynyl)-
benzoate (104). (S)-Methyl 3-bromo-4-(1-phenylethoxy)benzoate
(103, 3 g, 8.95 mmol) was taken up into triethylamine (60 mL, 430
mmol), and the solution was degassed with nitrogen for 30 min.
Copper(I) iodide (0.170 g, 0.895 mmol), trimethylsilylacetylene
(16.33 mL, 116 mmol), and Pd(PPh3)4 (1.034 g, 0.895 mmol) were
added, and the solution was stirred at 90 °C for 4 h. The reaction
mixture was filtered through celite and the filtrate was concentrated in
vacuo. The residue was partitioned between water and DCM (100 mL
each). The organic layer was eluted through a hydrophobic frit and
concentrated in vacuo. The resulting solid was purified by silica
chromatography eluting with 0−20% EtOAc/cyclohexane to give (S)-
methyl 4-(1-phenylethoxy)-3-((trimethylsilyl)ethynyl)benzoate (104,
3.75 g, 10.64 mmol, 119% yield). LC−MS (formic, ES+) tR = 1.61
min; m/z = 353; 1H NMR (400 MHz, CDCl3-d): δ ppm 8.12 (d, J =
2.4 Hz, 1H), 7.81−7.87 (m, 1H), 7.26−7.46 (m, 5H), 6.78 (d, J = 8.8
Hz, 1H), 5.48 (q, J = 6.4 Hz, 1H), 3.87 (s, 3H), 1.71 (d, J = 6.8 Hz,
3H), 0.25−0.33 (m, 9H).
(S)-Methyl 3-Ethynyl-4-(1-phenylethoxy)benzoate (105). (S)-
Methyl 4-(1-phenylethoxy)-3-((trimethylsilyl)ethynyl)benzoate
(104, 3.17 g, 8.99 mmol) was taken up into methanol (50 mL),
and potassium carbonate (3.73 g, 27.0 mmol) was added. The
reaction was stirred at room temperature for 1 h and then diluted with
water (100 mL). The reaction mixture was extracted with DCM (3 ×
50 mL). The combined organic phases were washed with 2 M
aqueous HCl and then eluted through a hydrophobic frit and
concentrated in vacuo. The resulting solid was purified by silica
chromatography eluting with 0−30% EtOAc/cyclohexane. The
product-containing fractions were concentrated in vacuo to give (S)-
methyl 3-ethynyl-4-(1-phenylethoxy)benzoate (105, 1.76 g, 6.28
mmol, 70% yield). LC−MS (formic, ES+) tR = 1.30 min; m/z =
281; 1H NMR (400 MHz, CDCl3-d): δ ppm 8.15 (d, J = 2.0 Hz, 1H),
7.84 (dd, J = 8.8, 2.0 Hz, 1H), 7.26−7.48 (m, 5H), 6.77 (d, J = 8.8
Hz, 1H), 5.47 (q, J = 6.4 Hz, 1H), 3.87 (s, 3H), 1.73 (d, J = 6.4 Hz,
3H).
(S)-Methyl 3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-
phenylethoxy)benzoate (106). (S)-Methyl 3-ethynyl-4-(1-
phenylethoxy)benzoate (105, 130 mg, 0.464 mmol), sodium azide
(121 mg, 1.855 mmol), copper(II) sulfate (14.80 mg, 0.093 mmol),
and copper powder (29.4 mg, 0.464 mmol) were dissolved in tert-
butanol (1 mL) and water (1 mL). Iodomethane (116 μL, 1.855
mmol) was added, and the reaction mixture was heated in the
microwave at 125 °C for 2.5 h. The reaction mixture was diluted with
water (5 mL) and extracted with EtOAc (5 mL × 2). The organic
layers were combined, eluted through a hydrophobic frit, and
concentrated in vacuo. The resulting residue was purified by silica
chromatography eluting with 0−70% EtOAc/cyclohexane. The
product-containing fractions were concentrated in vacuo to give (S)-
methyl 3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-phenylethoxy)-
benzoate (106, 90 mg, 0.267 mmol, 58% yield) as a yellow solid.
LC−MS (formic, ES+) tR = 1.14 min; m/z = 338.
General Procedure B. N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-
yl)-4-phenethoxybenzamide (19). N-Ethyl-4-hydroxy-3-(1-methyl-
1H-1,2,3-triazol-4-yl)benzamide (102, 210 mg, 0.853 mmol) was
divided equally between 3 scintillation vials, to which DMF (2 mL)
and (2-bromoethyl)benzene (55.2 mg, 0.298 mmol), (1-
bromopropyl)benzene (59.4 mg, 0.298 mmol), and 4-
(bromomethyl)tetrahydro-2H-pyran (53.4 mg, 0.298 mmol) were
added. To each was added potassium carbonate (82 mg, 0.597
mmol), and the reaction mixture was stirred at 50 °C for 2 h. The
temperature was increased to 50 °C for 3 h and then lowered to 50
°C, and the reaction mixture was stirred for 17 h. (2-Bromoethyl)-
benzene (55.2 mg, 0.298 mmol), (1-bromopropyl)benzene (59.4 mg,
0.298 mmol), and 4-(bromomethyl)tetrahydro-2H-pyran (53.4 mg,
0.298 mmol) were added to their respective reaction mixtures and
potassium carbonate (82 mg, 0.597 mmol) was added to each, the
temperature increased to 50 °C, and the reaction mixture was stirred
for 3 h, after which each was diluted with water and extracted into
DCM. The organic extracts of each were washed with 10% LiCl, dried
via a hydrophobic frit, and concentrated in vacuo. The residues were
taken up into 50% MeOH/DMSO and purified by formic MDAP, and
pure fractions were concentrated in vacuo to give N-ethyl-3-(1-
methyl-1H-1,2,3-triazol-4-yl)-4-phenethoxybenzamide (19, 38 mg,
0.108 mmol, 38% yield). LC−MS (formic, ES+) tR = 0.99 min; m/z
(S)-N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-
phenylethoxy)benzamide (17). (S)-Methyl 3-(1-methyl-1H-1,2,3-
triazol-4-yl)-4-(1-phenylethoxy)benzoate (106, 40 mg, 0.119 mmol),
DIBAL-Me3 (24.31 mg, 0.095 mmol), and ethanamine (0.059 mL,
0.119 mmol) were dissolved in THF (2 mL) and heated in a
microwave at 130 °C for 20 min. The reaction mixture was quenched
1
= 351.1; (100% pure) H NMR (400 MHz, MeOD-d4): δ ppm 8.55
(d, J = 2.4 Hz, 1H) 8.32 (br s, 1H) 7.82 (dd, J = 8.6, 2.2 Hz, 1H) 7.60
(s, 1H) 7.32−7.38 (m, 4H) 7.23−7.30 (m, 1H) 7.20 (d, J = 8.8 Hz,
1H) 4.51 (t, J = 6.6 Hz, 2H) 4.02 (s, 3H) 3.38−3.48 (m, 2H) 3.22 (t,
J = 6.4 Hz, 2H) 1.25 (t, J = 7.3 Hz, 3H).
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N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((tetrahydro-2H-
pyran-4-yl)methoxy)benzamide (20). General procedure B was
followed to give N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((tetra-
hydro-2H-pyran-4-yl)methoxy)benzamide (20, 23 mg, 0.067 mmol,
23% yield). LC−MS (formic, ES+) tR = 0.77 min; m/z = 345.2;
(100% pure) 1H NMR (400 MHz, MeOD-d4): δ ppm 8.57 (d, J = 2.4
Hz, 1H) 8.26−8.46 (m, 1H) 8.23 (s, 1H) 7.84 (dd, J = 8.8, 2.4 Hz,
1H) 7.20 (d, J = 8.8 Hz, 1H) 4.19 (s, 3H) 4.10 (d, J = 6.4 Hz, 2H)
4.01 (dd, J = 11.0, 3.2 Hz, 2H) 3.40−3.57 (m, 4H) 2.18−2.33 (m,
1H) 1.74−1.85 (m, 2H) 1.44−1.58 (m, 2H) 1.26 (t, J = 7.3 Hz, 3H).
Methyl 4-Hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoate,
Hydrochloride (65). Methyl 4-(methoxymethoxy)-3-(1-methyl-1H-
1,2,3-triazol-4-yl)benzoate (57, 15.7 g, 56.6 mmol) was dissolved in
DCM (10 mL) and 4 N HCl in 1,4-dioxane (70.8 mL, 283 mmol)
was added. The mixture was stirred at room temperature over the
weekend. The solvent was then evaporated in vacuo, and the residue
was triturated with ether (100 mL). The solid was collected by
filtration to give methyl 4-hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-
yl)benzoate, hydrochloride (65, 11.5 g, 42.6 mmol, 75% yield) as a
pale yellow solid. LC−MS (formic, ES+) tR = 0.60 min; m/z = 234;
1H NMR (400 MHz, DMSO-d6, 303 K): δ ppm 11.20 (bs, 1H), 8.69
1,2,3-triazol-4-yl)benzamide (102, 70 mg, 0.284 mmol), potassium
carbonate (118 mg, 0.853 mmol), and 4-(chloromethyl)pyridine,
hydrochloride (51.3 mg, 0.313 mmol) were stirred in acetone (10
mL) at 50 °C for 16 h. The reaction mixture was taken up into DMF
(5 mL) and 4-(chloromethyl)pyridine, hydrochloride (51.3 mg, 0.313
mmol) and potassium carbonate (118 mg, 0.853 mmol) were added,
and the reaction mixture was stirred at 50 °C for 3 h. The reaction
mixture was stirred at 50 °C for a further 3 h, after which stirring was
stopped and the reaction mixture was left at rt for 72 h. The solvent
was removed in vacuo and the residue was taken into water and
extracted into DCM. The organic extracts were combined and dried
via a hydrophobic frit and concentrated in vacuo. The residue was
taken up into 50% DMSO/MeOH (0.9 mL) and purified by formic
MDAP, and the relevant fractions were combined and concentrated to
give N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(pyridin-4-
ylmethoxy)benzamide (23, 71 mg, 0.210 mmol, 74% yield). LC−
MS (formic, ES+) tR = 0.45 min; m/z = 338.2; (100% pure) 1H NMR
(400 MHz, MeOD-d4): δ ppm 8.54−8.63 (m, 3H) 8.29 (s, 1H) 8.11
(s, 1H) 7.79 (dd, J = 8.6, 2.2 Hz, 1H) 7.53 (d, J = 6.4 Hz, 2H) 7.16
(d, J = 8.3 Hz, 1H) 5.45 (s, 2H) 4.17 (s, 3H) 3.43 (q, J = 7.0 Hz, 2H)
1.25 (t, J = 7.3 Hz, 3H).
(d, J = 2.4 Hz, 1H), 8.44 (s, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.12
(d, J = 8.8 Hz, 1H), 4.12 (s, 3H), 3.84 (s, 3H).
N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(pyrimidin-2-
ylmethoxy)benzamide (24). N-Ethyl-4-hydroxy-3-(1-methyl-1H-
1,2,3-triazol-4-yl)benzamide (102, 300 mg, 1.218 mmol) was
sonicated and heated to 70 °C and stirred to create a homologous
suspension. To a test tube, pyrimidin-2-ylmethanol (40.2 mg, 0.365
mmol) was added followed by tributylphosphine (0.122 mL, 0.487
mmol) and a quarter of the aforementioned suspension (5 mL). The
reaction mixture was cooled to 0 °C and ADDP (215 mg, 0.853
mmol) was added to each. The reaction mixture was warmed to 50 °C
and stirred for 17 h. The reaction mixture was concentrated in vacuo,
diluted with water, and extracted into DCM. The organic extracts
were combined, dried via a hydrophobic frit, and concentrated in
vacuo. The residue was dissolved in DCM and purified by silica
chromatography eluting 0−5% MeOH/DCM. The relevant fractions
were combined and concentrated, and the residues were then taken
up into 50% DMSO/MeOH (0.9 mL) and purified by formic MDAP
and the relevant fractions were combined and concentrated to give N-
ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(pyrimidin-2-ylmethoxy)-
benzamide (24, 35 mg, 0.103 mmol, 34% yield). LC−MS (formic,
ES+) tR = 0.67 min; m/z = 339.1; (100% pure) 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.84−8.93 (m, 2H) 8.68 (d, J = 2.4 Hz, 1H) 8.36
(br s, 1H) 7.80 (dd, J = 8.6, 2.2 Hz, 1H) 7.47 (t, J = 4.9 Hz, 1H) 7.23
(d, J = 8.8 Hz, 1H) 5.53 (s, 2H) 4.19 (s, 3H) 3.39−3.49 (m, 2H) 1.26
(t, J = 7.3 Hz, 3H).
(R)-3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-phenylethoxy)benzoic
Acid (108). NaOH (10 mL, 20.00 mmol) was added to a solution of
(S)-methyl 3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-phenylethoxy)-
benzoate (106, 1.5 g, 4.45 mmol) in methanol (20 mL) at room
temperature and the solution was stirred for 3 h and then evaporated
in vacuo. The residue was partitioned between water (20 mL) and
ether (20 mL). The aqueous layer was acidified with 2 M aqueous
HCl to pH 4 and stirred for 10 min. The resulting solid was collected
by filtration and dried in a vacuum oven to give (R)-3-(1-methyl-1H-
1,2,3-triazol-4-yl)-4-(1-phenylethoxy)benzoic acid (108, 0.75 g, 2.319
mmol, 52% yield) as a colorless solid. LC−MS (high pH, ES+) tR =
0.69 min; m/z = 324; 1H NMR (400 MHz, DMSO-d6): δ ppm 12.66
(br s, 1H), 8.75 (d, J = 2.4 Hz, 1H), 8.50 (s, 1H), 7.70 (dd, J = 8.8,
2.4 Hz, 1H), 7.40−7.50 (m, 2H), 7.24−7.38 (m, 3H), 7.07 (d, J = 8.8
Hz, 1H), 5.80 (q, J = 6.4 Hz, 1H), 4.19 (s, 3H), 1.74 (d, J = 6.4 Hz,
3H).
(R)-N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-
phenylethoxy)benzamide (21). (R)-3-(1-Methyl-1H-1,2,3-triazol-4-
yl)-4-(1-phenylethoxy)benzoic acid (108, 0.233 g, 0.72 mmol) and
HATU (0.274 g, 0.72 mmol) were dissolved in DMF (4.2 mL). A
total of 0.38 mL (2.16 mmol) of DIPEA was added to the solution
and the mixture was left for 5 min. A total of 0.76 mL (0.12 mmol
acid, 0.12 mmol HATU, 0.36 mmol DIPEA) of the mixture was
added to ethylamine (0.005 g, 0.120 mmol). The reaction mixture was
sealed and left stirring at 22 °C for 18 h. The reaction mixture was
purified by high pH MDAP, and the product-containing fractions
were concentrated under a stream of nitrogen to give (R)-N-ethyl-3-
(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-phenylethoxy)benzamide (21,
30.1 mg, 0.086 mmol, 64% yield). LC−MS (formic, ES+) tR = 0.98
N-Ethyl-4-((2-methoxybenzyl)oxy)-3-(1-methyl-1H-1,2,3-triazol-
4-yl)benzamide (25). A solution of N-ethyl-4-hydroxy-3-(1-methyl-
1H-1,2,3-triazol-4-yl)benzamide (102, 25 mg, 0.102 mmol) was
dissolved in DMF (0.5 mL) and added to 1-(chloromethyl)-2-
methoxybenzene (24 mg, 0.152 mmol). Potassium carbonate (28.1
mg, 0.203 mmol) was added. A stirrer bar was added and vials were
sealed and stood at room temperature for 18 h. More 1-
(chloromethyl)-2-methoxybenzene (excess 50 μL) and potassium
carbonate (28 mg) were added. The reaction vessels were sealed and
reheated in Anton Parr using initial 600W to 70 °C for 15 min. They
were reheated again in Anton Parr using initial 600W to 110 °C for 15
min. The sample was injected as is (filtered) and purified by high pH
MDAP. The solvent was dried under a stream of nitrogen in the plate
blowdown apparatus to give N-ethyl-4-((2-methoxybenzyl)oxy)-3-(1-
methyl-1H-1,2,3-triazol-4-yl)benzamide (25, 6 mg, 0.016 mmol, 15%
yield). LC−MS (formic, ES+) tR = 0.97 min; m/z = 367.0; (96%
1
min; m/z = 351; (100% pure) H NMR (600 MHz, DMSO-d6): δ
ppm 8.64 (d, J = 2.3 Hz, 1H), 8.49 (s, 1H), 8.38 (br t, J = 5.6 Hz,
1H), 7.62 (dd, J = 8.8, 2.4 Hz, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.34 (t, J
= 7.7 Hz, 2H), 7.14−7.29 (m, 1H), 7.02 (d, J = 8.7 Hz, 1H), 5.76−
5.81 (m, 1H), 4.19 (s, 3H), 3.25 (dt, J = 13.5, 6.6 Hz, 2H), 1.73 (d, J
= 6.4 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H).
N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(pyridin-3-
ylmethoxy)benzamide (22). General procedure B was followed to
give N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(pyridin-3-
ylmethoxy)benzamide (22, 46 mg, 0.136 mmol, 48% yield). LC−
MS (formic, ES+) tR = 0.49 min; m/z = 338.1; (100% pure) 1H NMR
(400 MHz, MeOD-d4): δ ppm 8.69 (d, J = 2.0 Hz, 1H) 8.60 (d, J =
2.4 Hz, 1H) 8.55 (dd, J = 4.9, 1.5 Hz, 1H) 8.32−8.41 (m, 1H) 8.16
(s, 1H) 7.98 (dt, J = 7.8, 1.7 Hz, 1H) 7.82 (dd, J = 8.8, 2.4 Hz, 1H)
7.50 (dd, J = 7.8, 4.9 Hz, 1H) 7.27 (d, J = 8.8 Hz, 1H) 5.41 (s, 2H)
4.11 (s, 3H) 3.38−3.48 (m, 2H) 1.25 (t, J = 7.1 Hz, 3H).
1
pure) H NMR (400 MHz, DMSO-d6): δ ppm 8.64 (d, J = 2.4 Hz,
1H) 8.44 (br t, J = 5.6 Hz, 1H) 8.23 (s, 1H) 7.78 (dd, J = 8.8, 2.4 Hz,
1H) 7.31−7.41 (m, 2H) 7.24 (d, J = 8.8 Hz, 1H) 7.09 (d, J = 9.3 Hz,
1H) 6.91−6.98 (m, 1H) 5.34 (s, 2H) 4.08 (s, 3H) 3.87 (s, 3H) 3.24−
3.29 (m, 2H) 1.08−1.15 (m, 3H).
N-Ethyl-4-((3-methoxybenzyl)oxy)-3-(1-methyl-1H-1,2,3-triazol-
4yl)benzamide (26). General procedure B was followed using silica
chromatography eluting with EtOAc to give N-ethyl-4-((3-
methoxybenzyl)oxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzamide
N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(pyridin-4-
ylmethoxy)benzamide (23). N-Ethyl-4-hydroxy-3-(1-methyl-1H-
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(26, 142 mg, 0.388 mmol, 77% yield) as a colorless oil. LC−MS
The residue was taken into MeOH and passed through an Isolute
NH2 ion-exchange column. The eluent was concentrated in vacuo, and
the residue was stirred in DCM, sonicated, and heated to reach
complete dissolution. This was then purified by silica chromatography
eluting with 0−5% MeOH/DCM. The relevant fractions were
combined and concentrated to give rac-methyl 3-(1-methyl-1H-
1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoate (900 mg, 2.66
mmol, 76% yield) as a white solid. The racemic mixture was dissolved
in EtOH (9 mL) and injected onto the column (column: 30 mm × 25
cm Chiralpak AD-H, 5 μm), eluting with 25% EtOH/heptane, flow
rate = 30 mL/min, detection wavelength 215.4 nm. The pure fractions
from peak 1 were combined and concentrated in vacuo to give rel-(R)-
methyl 3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
benzoate (59, 377 mg, 1.114 mmol, 32% yield). LC−MS (formic,
ES+) tR = 0.86 min; m/z = 339.1; 1H NMR (400 MHz, DMSO-d6): δ
ppm 8.78 (d, J = 2.4 Hz, 1H) 8.59−8.63 (m, 1H) 8.57 (s, 1H) 7.73−
7.82 (m, 2H) 7.41 (d, J = 7.8 Hz, 1H) 7.32 (ddd, J = 7.5, 4.8, 1.0 Hz,
1H) 7.07 (d, J = 8.8 Hz, 1H) 5.79 (q, J = 6.4 Hz, 1H) 4.17 (s, 3H)
3.83 (s, 3H) 1.77 (d, J = 6.4 Hz, 3H).
1
(formic, ES+) tR = 0.95; m/z = 367.1; (100% pure) H NMR (400
MHz, MeOD-d4): δ ppm 8.61 (d, J = 2.4 Hz, 1H) 8.19 (s, 1H) 7.79
(dd, J = 8.8, 2.4 Hz, 1H) 7.32 (t, J = 8.1 Hz, 1H) 7.21 (d, J = 8.3 Hz,
1H) 7.01−7.07 (m, 2H) 6.88−6.95 (m, 1H) 5.30 (s, 2H) 4.81 (s,
3H) 4.11 (s, 3H) 3.43 (q, J = 7.3 Hz, 2H) 1.25 (t, J = 7.1 Hz, 3H).
N-Ethyl-4-((4-methoxybenzyl)oxy)-3-(1-methyl-1H-1,2,3-triazol-
4-yl)benzamide (27). General procedure B was followed using silica
chromatography eluting with EtOAc to give N-ethyl-4-((4-
methoxybenzyl)oxy)-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzamide
(27, 121 mg, 0.330 mmol, 66% yield) as a white solid. LC−MS
1
(formic, ES+) tR = 0.94 min; m/z = 367.1; (100% pure) H NMR
(400 MHz, MeOD-d4): δ ppm 8.62 (d, J = 2.4 Hz, 1H) 8.10 (s, 1H)
7.77−7.83 (m, 1H) 7.37−7.45 (m, 2H) 7.24 (d, J = 8.8 Hz, 1H)
6.94−7.00 (m, 2H) 5.24 (s, 2H) 4.82 (s, 3H) 4.08 (s, 3H) 3.43 (q, J
= 7.3 Hz, 2H) 1.25 (t, J = 7.3 Hz, 3H).
N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((6-methylpyridin-2-
yl)methoxy)benzamide (28). General procedure B was followed to
give N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((6-methylpyridin-
2-yl)methoxy)benzamide (28, 51 mg, 0.145 mmol, 60% yield).
Rel-(R)-3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)benzoic Acid (66). LiOH (110 mg, 4.59 mmol) and rel-(R)-
methyl 3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
benzoate (59, 377 mg, 1.114 mmol) were stirred in THF (5 mL) and
water (5 mL) at 50 °C for 16 h. The reaction mixture was
concentrated to about half volume in vacuo and 2 M HCl was added
dropwise until a white precipitate formed. The suspension was then
filtered through a sinter funnel; however, this did not successfully
isolate the precipitate. The sinter funnel was washed with ethyl
acetate, and the suspension was extracted with EtOAc (2 × 10 mL).
The organics were combined, dried via a hydrophobic frit, and
concentrated in vacuo to give a clear glassy residue. LC−MS of the
aqueous phase suggested that the product remained and so 2 M HCl
was added until the addition of a drop caused resolvation of the
product (due to zwitterionic nature of product). A drop of 2 M
NaOH was then added and the pH was tested as pH 2. The aqueous
fraction was extracted again with EtOAc (2 × 10 mL), and the
organics were combined, dried via a hydrophobic frit, combined with
the previous residue, and concentrated in vacuo to give rel-(R)-3-(1-
methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoic acid
(66, 254 mg, 0.783 mmol, 70% yield) as a white solid. LC−MS
1
LC−MS (formic, ES+) tR = 0.58 min; m/z = 352.1; (94% pure) H
NMR (400 MHz, MeOD-d4): δ ppm 8.63 (d, J = 2.4 Hz, 1H) 8.41 (s,
1H) 8.33−8.38 (m, 1H) 7.80 (dd, J = 8.8, 2.4 Hz, 1H) 7.75 (t, J = 7.8
Hz, 1H) 7.34 (d, J = 7.8 Hz, 1H) 7.28 (d, J = 7.3 Hz, 1H) 7.20 (d, J =
8.8 Hz, 1H) 5.39 (s, 2H) 4.15 (s, 3H) 3.39−3.48 (m, 2H) 2.60 (s,
3H) 1.25 (t, J = 7.1 Hz, 3H).
Ethyl 4-Hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoate
(108). 4-Hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoic acid
(97, 900 mg, 4.11 mmol), concn sulfuric acid (0.1 mL, 1.876
mmol), and ethanol (10 mL) were stirred at 50 °C for 16 h. The
reaction mixture was stirred at 80 °C for 24 h. The reaction mixture
was concentrated and dried to give ethyl 4-hydroxy-3-(1-methyl-1H-
1,2,3-triazol-4-yl)benzoate (108, 890 mg, 3.60 mmol, 88% yield) as a
1
cream solid. LC−MS (formic, ES+) tR = 0.91 min; m/z = 248.1; H
NMR (400 MHz, DMSO-d6): δ ppm 11.06 (br s, 1H) 8.66−8.71 (m,
1H) 8.44 (s, 1H) 7.76−7.83 (m, 1H) 7.06 (d, J = 8.3 Hz, 1H) 4.12 (s,
3H) 3.75 (q, J = 7.0 Hz, 2H) 1.08−1.14 (m, 3H).
Ethyl 3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)benzoate (109). General procedure B was followed with
heating at 65 °C and using silica chromatography to give ethyl 3-(1-
methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoate
(109, 1.36 g, 3.86 mmol, 81% yield). LC−MS (formic, ES+) tR = 0.95
min; m/z = 353.1.
1
(formic, ES+) tR = 0.69 min; m/z = 325.1; H NMR (400 MHz,
DMSO-d6): δ ppm 12.63 (br s, 1H) 8.76 (d, J = 2.4 Hz, 1H) 8.59−
8.63 (m, 1H) 8.55 (s, 1H) 7.70−7.82 (m, 2H) 7.38−7.43 (m, 1H)
7.28−7.36 (m, 1H) 7.03 (d, J = 8.8 Hz, 1H) 5.77 (q, J = 6.4 Hz, 1H)
4.17 (s, 3H) 1.77 (d, J = 6.4 Hz, 3H).
3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
benzoic Acid (110). Ethyl 3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-
(pyridin-2-yl)ethoxy)benzoate (109, 1.36 g, 3.86 mmol) was stirred
with LiOH (370 mg, 15.44 mmol) in THF (20 mL) and water (20
mL) at 50 °C for 16 h. Approximately, half the solvent was removed
in vacuo and the mixture was acidified with 2 M HCl to pH 6, giving a
white precipitate. This was isolated by filtration to give 3-(1-methyl-
1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoic acid (110,
1.07 g, 3.30 mmol, 85% yield) as a white solid. LC−MS (formic,
ES+) tR = 0.71 min; m/z = 325.1; 1H NMR (400 MHz, DMSO-d6): δ
ppm 12.69 (br s, 1H) 8.76 (d, J = 2.4 Hz, 1H) 8.57−8.62 (m, 1H)
8.55 (s, 1H) 7.71−7.82 (m, 2H) 7.36−7.44 (m, 1H) 7.25−7.36 (m,
1H) 7.03 (d, J = 9.3 Hz, 1H) 5.76 (q, J = 6.4 Hz, 1H) 4.17 (s, 3H)
1.76 (d, J = 6.4 Hz, 3H).
Rel-(R)-Methyl 3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-
yl)ethoxy)benzoate (59). 3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-
(pyridin-2-yl)ethoxy)benzoic acid (110, 1.13 g, 3.48 mmol) was
stirred in methanol (20 mL) and hydrochloric acid (0.106 mL, 3.48
mmol) at 100 °C for 2 h. Hydrochloric acid (0.212 mL, 6.97 mmol)
was added, and the reaction mixture was stirred at 100 °C for 16 h.
The reaction mixture was stirred at 100 °C for a further 24 h.
Hydrochloric acid (1 mL) was added, and the reaction mixture was
stirred at 100 °C for 24 h. Hydrochloric acid (1 mL) was added, and
the reaction mixture was stirred at 100 °C for 24 h. The reaction
mixture was concentrated in vacuo, partitioned between water and
EtOAc, and extracted into EtOAc. The organic extracts were
combined, dried via a hydrophobic frit, and concentrated in vacuo.
General Procedure C. In four vials, HATU (164 mg, 0.431
mmol) was weighed in each. (S)-3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-
(1-(pyridin-2-yl)ethoxy)benzoic acid (254 mg, 0.783 mmol) was
dissolved in DMF (6 mL) and dispensed in portions (1.5 mL) to each
vial. To each vial was added DIPEA (0.075 mL, 0.431 mmol), and the
reaction mixtures were stirred for 15 min at rt. Amine (0.230 mmol)
was then added. The reaction mixtures were stirred for 64 h. The
reaction mixtures were diluted with water (5 mL) and saturated
sodium bicarbonate solution (3 mL) and extracted into DCM (2 × 10
mL). The organics were washed with an equal volume of 10% LiCl
aqueous solution, dried via a hydrophobic frit, and concentrated in
vacuo. The residues were dissolved in 50% MeOH/DMSO (0.9 mL)
and purified by MDAP (formic acid method), and the relevant
fractions were combined to give the product.
(S)-N-Ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)benzamide (29). General procedure C was followed to give
(S)-N-ethyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)benzamide (29, 50 mg, 0.142 mmol, 73% yield). LC−MS
1
(formic, ES+) tR = 0.69 min; m/z = 352.1; (100% pure) H NMR
(400 MHz, MeOD-d4): δ ppm 8.54−8.65 (m, 2H) 8.49 (s, 1H) 7.80
(td, J = 7.7, 1.7 Hz, 1H) 7.64 (dd, J = 8.8, 2.4 Hz, 1H) 7.45 (d, J = 7.8
Hz, 1H) 7.35 (ddd, J = 7.5, 5.0, 1.2 Hz, 1H) 6.94 (d, J = 8.8 Hz, 1H)
5.69 (q, J = 6.5 Hz, 1H) 4.22 (s, 3H) 3.40 (q, J = 7.3 Hz, 2H) 1.82 (d,
J = 6.4 Hz, 3H) 1.22 (t, J = 7.3 Hz, 3H).
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1
tert-Butyl 2-Chloro-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (69). General procedure A was followed, purified by
silica chromatography eluting with 0−70% EtOAc/cyclohexane, to
give tert-butyl 2-chloro-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (69, 1.8046 g, 6.12 mmol, 89% yield) as a pale orange
(formic, ES+) tR = 0.94 min; m/z = 340.2; (100% pure) H NMR
(400 MHz, CDCl3-d): δ ppm 8.15−8.19 (m, 2H) 7.56 (d, J = 1.5 Hz,
1H) 7.22−7.30 (m, 1H) 7.07 (d, J = 7.3 Hz, 1H) 7.01 (dt, J = 10.0,
1.8 Hz, 1H) 6.92 (td, J = 8.4, 2.7 Hz, 1H) 6.35 (br s, 1H) 4.13−4.23
(m, 5H) 3.51 (qd, J = 7.3, 5.6 Hz, 2H) 1.27 (obs. t, J = 7.3 Hz, 3H).
N-Ethyl-2-(4-fluorobenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (35). General procedure D was followed to give N-
ethyl-2-(4-fluorobenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (35, 46.9 mg, 0.138 mmol, 55% yield). LC−MS
(formic, ES+) tR = 0.93 min; m/z = 340.2; (85% pure) 1H NMR (400
MHz, CDCl3-d): δ ppm 8.13−8.19 (m, 2H) 7.54 (d, J = 1.5 Hz, 1H)
7.22−7.30 (m, 4H) 6.94−7.03 (m, 2H) 6.36 (br s, 1H) 4.14−4.24
(m, 5H) 3.47−3.55 (m, 2H) 1.27 (t, J = 7.3 Hz, 3H).
1
solid. LC−MS (formic, ES+) tR = 1.18 min; m/z = 295.1; H NMR
(400 MHz, CDCl3-d): δ ppm 8.55 (d, J = 1.5 Hz, 1H) 8.20 (s, 1H)
7.75−7.79 (m, 1H) 4.19 (s, 3H) 1.64 (s, 9H).
General Procedure D. 2-Chloro-N-ethyl-6-(1-methyl-1H-1,2,3-
triazol-4-yl)isonicotinamide (71, 60 mg, 0.226 mmol) and
PdCl2(PPh3)2 (16 mg, 0.023 mmol) were dissolved in THF (2 mL)
in a microwave vial. The vial was sealed and Negishi reagent (0.5 M in
THF) (0.677 mL, 0.339 mmol) was added. The vial was heated to
110 °C for 1 h. The reaction mixture was blown down under a stream
of nitrogen and the resulting solid was dissolved in 1:1 DMSO/
methanol and purified by formic MDAP. The product-containing
fractions were concentrated in vacuo, and the product was left to dry
under a stream of nitrogen overnight to give the required products
listed below.
Methyl 2-Methyl-6-((trimethylsilyl)ethynyl)isonicotinate (111). A
solution of methyl 2-bromo-6-methylisonicotinate (30 g, 130 mmol)
and Et3N (182 mL, 1304 mmol) was degassed with nitrogen for 10
min followed by the addition of Pd(Ph3P)4 (7.53 g, 6.52 mmol),
copper(I) iodide (2.484 g, 3.04 mmol), and ethynyltrimethylsilane
(111 mL, 782 mmol) and again degassed with nitrogen for 5 min. The
reaction mixture was heated at 90 °C for 3 h and then cooled and
filtered. Water (200 mL) was added to the filtrate and it was extracted
with ethyl acetate (200 mL × 2). The combined organic layers were
dried with Na2SO4, filtered, and concentrated in vacuo to give methyl
2-methyl-6-((trimethylsilyl)ethynyl)isonicotinate (111, 38 g, 82
mmol, 72% yield). LC−MS (formic, ES+) tR = 2.00 min; m/z =
248; 1H NMR (400 MHz, CDCl3-d): δ ppm 7.82 (s, 1H) 7.64 (obs. s,
1H) 3.94 (s, 3H) 2.62 (s, 3H) 0.25−0.32 (m, 9H).
N-Ethyl-2-(2-methoxybenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (30). General procedure D was followed including
an additional purification by silica chromatography eluting with 30−
100% EtOAc/cyclohexane to give N-ethyl-2-(2-methoxybenzyl)-6-(1-
methyl-1H-1,2,3-triazol-4-yl)isonicotinamide (30, 11.5 mg, 0.033
mmol, 14% yield). LC−MS (formic, ES+) tR = 0.92 min; m/z =
1
352.2; (100% pure) H NMR (400 MHz, CDCl3-d): δ ppm 8.16 (s,
1H) 8.13 (d, J = 2.0 Hz, 1H) 7.51 (d, J = 1.5 Hz, 1H) 7.24 (dt, J =
7.3, 1.5 Hz, 1H) 7.19 (dd, J = 8.1, 1.7 Hz, 1H) 6.87−6.93 (m, 2H)
6.30 (br s, 1H) 4.22 (s, 2H) 4.18 (s, 3H) 3.82 (s, 3H) 3.45−3.54 (m,
2H) 1.26 (t, J = 7.3 Hz, 3H).
Methyl 2-Methyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate
(84). Methyl 2-methyl-6-((trimethylsilyl)ethynyl)isonicotinate (111,
30 g, 74.5 mmol), ascorbic acid (6.56 g, 37.3 mmol), sodium azide
(10.17 g, 156 mmol), copper(II) sulfate (2.379 g, 14.90 mmol), and
K2CO3 (15.45 g, 112 mmol) were combined in water (50 mL) and
methanol (50.0 mL). The reaction mixture was stirred at room
temperature. MeI (14.91 mL, 238 mmol) and pyridine (30.1 mL, 373
mmol) were added and the reaction mixture was stirred at room
temperature until no starting material remained by TLC (30% EtOAc
in pet ether). The reaction mixture was concentrated under reduced
pressure to remove the methanol. The mixture was diluted with water
(200 mL) and extracted with ethyl acetate (300 mL × 3). The
combined organic extracts were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was triturated with
10% ethyl acetate in pet ether (50 mL), and the resulting solid was
dried to give methyl 2-methyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (84, 20 g, 59.6 mmol, 80% yield). LC−MS (formic,
N-Ethyl-2-(3-methoxybenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (31). General procedure D was followed using the
following amounts: 0.5 M (3-methoxybenzyl)zinc(II) chloride in
THF (4.35 mL, 2.175 mmol), 2-chloro-N-ethyl-6-(1-methyl-1H-1,2,3-
triazol-4-yl)isonicotinamide (71, 289 mg, 1.088 mmol) and
PdCl2(PPh3)2 (153 mg, 0.218 mmol) in THF (7 mL), and was
purified by silica chromatography using a gradient of 33−100% ethyl
acetate/cyclohexane. The product-containing fractions were com-
bined, and the solvent was removed in vacuo. The product was left to
dry in vacuo for 1 h to give N-ethyl-2-(3-methoxybenzyl)-6-(1-methyl-
1H-1,2,3-triazol-4-yl)isonicotinamide (31, 171 mg, 0.487 mmol, 45%
yield) as a pale brown solid. LC−MS (formic, ES+) tR = 0.90 min; m/
z = 352; (100% pure) 1H NMR (400 MHz, CDCl3-d): δ ppm 8.18 (s,
1H), 8.17 (d, J = 1.5 Hz, 1H), 7.56 (d, J = 1.5 Hz, 1H), 7.21−7.26
(m, 1H), 6.88−6.92 (m, 1H), 6.85−6.87 (m, 1H), 6.77−6.81 (m,
1H), 6.25−6.33 (m, 1H), 4.18−4.21 (m, 5H), 3.80 (s, 3H), 3.47−
3.56 (m, 3H), 1.28 (t, J = 7.3 Hz, 3H).
ES+) tR = 1.97 min; m/z = 233; H NMR (400 MHz, DMSO-d6): δ
ppm 8.64 (s, 1H) 8.25 (s, 1H) 7.68 (obs. s, 1H) 4.13 (s, 3H) 3.93 (s,
3H) 2.60 (s, 3H).
1
4-(Methoxycarbonyl)-2-methyl-6-(1-methyl-1H-1,2,3-triazol-4-
yl)pyridine-1-oxide (85). To a solution of methyl 2-methyl-6-(1-
methyl-1H-1,2,3-triazol-4-yl)isonicotinate (84, 15 g, 44.6 mmol) in
DCM (100 mL) was added a solution of 3-chlorobenzoperoxoic acid
(15.38 g, 89 mmol). The reaction mixture was stirred at room
temperature for 18 h. The reaction mixture was concentrated under
reduced pressure to remove the DCM. Water (100 mL) was added to
the reaction mixture, and the reaction mixture was neutralized with aq
saturated NaHCO3. The mixture was extracted with ethyl acetate (3 ×
200 mL). The combined organic extracts were dried over Na2OSO4,
filtered, and concentrated under reduced pressure. The residue was
triturated with diethyl ether (200 mL), and the resulting solid was
collected by filtration to give 4-(methoxycarbonyl)-2-methyl-6-(1-
methyl-1H-1,2,3-triazol-4-yl)pyridine 1-oxide (85, 12 g, 43.5 mmol,
N-Ethyl-2-(4-methoxybenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (32). General procedure D was followed including
an additional purification by silica chromatography eluting with 30−
100% EtOAc/cyclohexane to give N-ethyl-2-(4-methoxybenzyl)-6-(1-
methyl-1H-1,2,3-triazol-4-yl)isonicotinamide (32, 15.7 mg, 0.045
mmol, 19% yield). LC−MS (formic, ES+) tR = 0.90 min; m/z =
1
352.2; (100% pure) H NMR (400 MHz, CDCl3-d): δ ppm 8.17 (s,
1H) 8.14 (d, J = 1.5 Hz, 1H) 7.52 (d, J = 1.5 Hz, 1H) 7.21 (qt, J =
8.3, 1.5 Hz, 2H) 6.85 (qt, J = 8.8, 2.9 Hz, 2H) 6.32 (br s, 1H) 4.19 (s,
3H) 4.15 (s, 2H) 3.79 (s, 3H) 3.45−3.55 (m, 2H) 1.26 (t, J = 7.1 Hz,
3H).
N-Ethyl-2-(2-fluorobenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (33). General procedure D was followed to give N-
ethyl-2-(2-fluorobenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (33, 21 mg, 0.062 mmol, 26% yield). LC−MS
1
98% yield). LC−MS (formic, ES+) tR = 1.84 min; m/z = 249; H
1
(formic, ES+) tR = 0.92 min; m/z = 340.2; (100% pure) H NMR
NMR (400 MHz, DMSO-d6): δ ppm 9.12 (s, 1H) 8.71 (d, J = 2.4 Hz,
1H) 7.98 (d, J = 2.2 Hz, 1H) 4.17 (s, 3H) 3.92 (s, 3H) 3.30 (obs. s,
3H).
Methyl 2-(Hydroxymethyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (112). A solution of 4-(methoxycarbonyl)-2-methyl-6-
(1-methyl-1H-1,2,3-triazol-4-yl)pyridine 1-oxide (85, 11.5 g, 41.7
mmol) in TFAA (17.67 mL, 125 mmol) was stirred at 50 °C for 12 h.
The reaction mixture was quenched slowly with methanol and then
(400 MHz, CDCl3-d): δ ppm 8.12−8.21 (m, 2H) 7.55 (d, J = 1.5 Hz,
1H) 7.20−7.26 (m, 2H) 7.02−7.12 (m, 2H) 6.35 (br s, 2H) 4.25 (s,
2H) 4.18 (s, 3H) 3.46−3.55 (m, 2H) 1.27 (obs. t, J = 7.3 Hz, 3H).
N-Ethyl-2-(3-fluorobenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (34). General procedure D was followed to give N-
ethyl-2-(3-fluorobenzyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (34, 35 mg, 0.103 mmol, 44% yield). LC−MS
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concentrated under reduced pressure. The crude compound was
triturated with diethyl ether (50 mL), and the collected solid was
triturated with MeOH (50 mL). The resulting solid was collected by
filtration and dried to give methyl 2-(hydroxymethyl)-6-(1-methyl-
1H-1,2,3-triazol-4-yl)isonicotinate (112, 7.4 g, 28.5 mmol, 68% yield).
J = 7.6 Hz, 1H), 6.75−7.00 (m, 2H), 6.45−6.59 (m, 1H), 4.43 (s,
2H), 4.14 (s, 3H).
2-((1H-Indol-4-yl)methyl)-N-ethyl-6-(1-methyl-1H-1,2,3-triazol-
4-yl)isonicotinamide (36). General procedure C was followed using
the following amounts: HATU (151 mg, 0.396 mmol), 2-((1H-indol-
4-yl)methyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic acid (88
mg, 0.264 mmol), 2 M ethanamine in THF (0.172 mL, 0.343 mmol),
DIPEA (0.092 mL, 0.528 mmol), and DMF (4 mL). The sample was
purified using silica chromatography eluting with 50−80% EtOAc/
cyclohexane. The product-containing fractions were combined, and
the solvent was removed in vacuo to give 2-((1H-indol-4-yl)methyl)-
N-ethyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinamide (36, 41
mg, 0.102 mmol, 39% yield) as a white solid. LC−MS (formic,
1
LC−MS (formic, ES+) tR = 1.36 min; m/z = 249; H NMR (400
MHz, DMSO-d6): δ ppm 8.60 (s, 1H) 8.31 (br s, 1H) 7.88 (br s, 1H)
4.67 (s, 2H) 4.12 (obs. s, 3H) 3.95 (obs. s, 3H).
Methyl 2-(Chloromethyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (86). Thionyl chloride (2 mL, 27.4 mmol) was added
to methyl 2-(hydroxymethyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (112, 1.5 g, 6.04 mmol), and the reaction mixture was
heated to 90 °C under nitrogen for 25 min and then cooled to room
temperature. Ice (20 g) and sodium acetate (6 g) were added, and the
aqueous layer was extracted with ethyl acetate (3 × 30 mL). The
organic layer was filtered, and the filtrate was passed through a
hydrophobic frit and then concentrated in vacuo. The resulting solid
was purified by silica chromatography using a gradient of 20−80%
ethyl acetate/cyclohexane. The product-containing fractions were
combined, and the solvent was removed in vacuo to give methyl 2-
(chloromethyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate (86,
1.051 g, 3.94 mmol, 65.2% yield) as a pale yellow solid. LC−MS
(formic, ES+) tR = 0.86 min; m/z = 267; 1H NMR (400 MHz, CDCl3-
d): δ ppm 8.65 (d, J = 1.5 Hz, 1H), 8.19 (s, 1H), 7.97 (d, J = 1.0 Hz,
1H), 4.74 (s, 2H), 4.13−4.27 (m, 3H), 3.98−4.04 (m, 3H).
1
ES+) tR = 0.85 min; m/z = 361; (100% pure) H NMR (400 MHz,
DMSO-d6): δ ppm 11.08 (br s, 1H), 8.71−8.97 (m, 1H), 8.61 (s,
1H), 8.23 (d, J = 1.5 Hz, 1H), 7.51 (d, J = 1.5 Hz, 1H), 7.17−7.40
(m, 2H), 6.98−7.16 (m, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.36−6.63 (m,
1H), 4.40 (s, 2H), 4.14 (s, 3H), 3.17−3.28 (m, 2H), 1.05−1.22 (t,
3H).
(S)-Methyl 3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)benzoate (59). To a mixture of (R)-1-(pyridin-2-yl)ethanol
(2.8188 g, 22.89 mmol) and methyl 4-hydroxy-3-(1-methyl-1H-1,2,3-
triazol-4-yl)benzoate (114, 4.8116 g, 20.63 mmol) in toluene (100
mL) in a 250 mL round-bottomed flask was added 2-
(tributylphosphoranylidene)acetonitrile (16 mL, 61.1 mmol). The
mixture was stirred at 95 °C for 45 min. The reaction mixture was
then stirred at reflux for 4 h after which the reaction mixture was
removed from heating and cooled overnight. The mixture was
evaporated in vacuo and redissolved in DCM (10 mL). This solution
was split equally between two 100 g silica columns and was purified
by flash chromatography. Each column was eluted with a gradient of
30−90% EtOAc/cyclohexane, producing 2 collections for each
column of varying purities of the product. The first collections for
each column were combined, and the second collections for each
column were combined. These 2 combined solutions were both
separately concentrated in vacuo, before being dissolved in DCM (10
and 5 mL). Both solutions were repurified separately via normal-phase
column chromatography, eluting with a gradient of 30−90% EtOAc/
cyclohexane. All product-containing fractions were concentrated in
vacuo, dissolved in DCM (10 mL), and combined. The combined
material was concentrated in vacuo to give (S)-methyl 3-(1-methyl-
1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoate (59, 5.1842
g, 15.32 mmol, 74% yield) as a sticky brown solid. LC−MS (formic,
Methyl 2-((1H-Indol-4-yl)methyl)-6-(1-methyl-1H-1,2,3-triazol-4-
yl)isonicotinate (87). (1H-Indol-4-yl)boronic acid (543 mg, 3.37
mmol) was added to a solution of methyl 2-(chloromethyl)-6-(1-
methyl-1H-1,2,3-triazol-4-yl)isonicotinate (86, 750 mg, 2.81 mmol),
XPhos Pd G1 (104 mg, 0.141 mmol), and K2CO3 (800 mg, 5.79
mmol) in ethanol (2.222 mL) and toluene (20 mL). The reaction
mixture was stirred at 90 °C for 1.5 h and then an additional 1 equiv.
of XPhos PdG1 was added to the reaction mixture. The reaction
mixture was stirred for a further 20 h and then cooled to room
temperature. The reaction flask was degassed with nitrogen and an
additional equivalent of XPhos Pd G1 was added. The reaction
mixture was stirred for 2 h and Xphos (104 mg) was added. The
reaction mixture was stirred for 1 h at 90 °C and then cooled to room
temperature. The reaction mixture was partitioned between EtOAc
(25 mL) and water (30 mL). The organic layer was washed (1× water
20 mL, 2× sat. aq NaHCO3 20 mL), passed through a hydrophobic
frit, and concentrated in vacuo. The resulting solid was purified by
column chromatography using a gradient of 50−100% EtOAc/
cyclohexane. The product-containing fractions were combined, and
the solvent was removed in vacuo. The sample was then dried under a
stream of nitrogen for 1 h to give methyl 2-((1H-indol-4-yl)methyl)-
6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate (87, 190 mg, 0.273
mmol, 10% yield) as a white/brown solid. LC−MS (formic, ES+) tR =
0.99 min; m/z = 348; 1H NMR (400 MHz, DMSO-d6): δ ppm 11.09
(br s, 1H), 8.67 (s, 1H), 8.17−8.35 (m, 2H), 7.48−7.60 (m, 1H),
7.20−7.41 (m, 3H), 7.05 (dd, J = 8.1, 7.1 Hz, 2H), 6.84−6.99 (m,
2H), 6.43−6.59 (m, 1H), 4.45 (s, 2H), 4.17 (s, 3H), 3.86 (s, 3H).
2-((1H-Indol-4-yl)methyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinic Acid (113). Methyl 2-((1H-indol-4-yl)methyl)-6-(1-
methyl-1H-1,2,3-triazol-4-yl)isonicotinate (87, 203 mg, 0.292
mmol) and LiOH (28.0 mg, 1.169 mmol) were dissolved in water
(3 mL) and THF (3.00 mL). The reaction mixture was stirred for 1.5
h at 50 °C under nitrogen. The reaction mixture was concentrated in
vacuo to remove any THF. The aqueous solution remaining was
acidified using 2 M HCl and then concentrated in vacuo. The resulting
solid was purified by reverse-phase chromatography eluting with 5−
95% MeCN/0.1% formic acid in water. The product-containing
fractions were combined, and the solvent was removed in vacuo. The
sample was then dried for 14 h under a stream of nitrogen to give 2-
((1H-indol-4-yl)methyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinic acid (113, 62 mg, 0.167 mmol, 57% yield) as a green/
gray solid. LC−MS (formic, ES+) tR = 0.56 min; m/z = 334; 1H NMR
(400 MHz, DMSO-d6): δ ppm 11.10 (br s, 1H), 8.65 (s, 1H), 8.23 (d,
J = 1.0 Hz, 1H), 7.52 (d, J = 1.5 Hz, 1H), 7.24−7.35 (m, 2H), 7.05 (t,
1
ES+) tR = 0.86 min; m/z = 339.2; H NMR (400 MHz, CDCl3-d): δ
9.04 (d, J = 2.4 Hz, 1H) 8.64 (dt, J = 4.9, 1.0 Hz, 1H) 8.22 (s, 1H)
7.85 (dd, J = 8.8, 2.4 Hz, 1H) 7.63 (td, J = 7.8, 2.0 Hz, 1H) 7.26−7.20
(m, 2H) 6.85 (d, J = 8.8 Hz, 1H) 5.67 (q, J = 6.8 Hz, 1H) 4.21 (s,
3H) 3.90 (s, 3H) 1.84 (d, J = 6.4 Hz, 3H).
(S)-3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
benzoic Acid (66). To a mixture of (S)-methyl 3-(1-methyl-1H-1,2,3-
triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoate (59, 5.1788 g, 15.31
mmol) in water (60 mL) and THF (60 mL) in a 250 mL round-
bottomed flask was added lithium hydroxide (1.8546 g, 77 mmol).
This mixture was stirred at room temperature for 20.5 h. The reaction
mixture was washed with diethyl ether (3 × 100 mL), and the
aqueous layer was concentrated in vacuo. The mixture was acidified to
pH 3 using 2 M HCl and left to precipitate out for 48 h to give a
brown gum. The acidic mother liquor was decanted from the gum and
the gum was washed with water (2 × 20 mL). The gum was dried in
vacuo, triturated with diethyl ether (200 mL), and filtered. The solid
was dried in vacuo to give (S)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-
(pyridin-2-yl)ethoxy)benzoic acid (66, 3.2319 g, 9.96 mmol, 65%
yield) as a brown powder. LC−MS (formic, ES+) tR = 0.69 min; m/z
= 325.2; 1H NMR (400 MHz, DMSO-d6): δ 12.70 (br s, 1H) 8.76 (d,
J = 2.0 Hz, 1H) 8.60 (d, J = 4.9 Hz, 1H) 8.56 (s, 1H), 7.81−7.74 (m,
2H) 7.41 (d, J = 7.8 Hz, 1H) 7.33 (ddd, J = 7.3, 4.9, 1.0 Hz, 1H) 7.03
(d, J = 8.8 Hz, 1H) 5.77 (q, J = 6.7 Hz, 1H) 4.18 (s, 3H), 1.77 (d, J =
6.4 Hz, 3H).
3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)ethoxy)-
N-(2-(tetrahydro-2H-pyran-3-yl)ethyl)benzamide (37). General pro-
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1
cedure C was followed using the following amounts: (S)-3-(1-methyl-
1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoic acid (66, 90
mg, 0.277 mmol), DMF (2.5 mL), DIPEA (0.097 mL, 0.555 mmol),
HATU (127 mg, 0.333 mmol), and 2-(tetrahydro-2H-pyran-3-
yl)ethanamine (39.4 mg, 0.305 mmol). This gave 3-(1-methyl-1H-
1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)ethoxy)-N-(2-(tetrahydro-
2H-pyran-3-yl)ethyl)benzamide (37, 12.5 mg, 0.029 mmol, 10%
yield). LC−MS (formic, ES+) tR = 0.87 min; m/z = 436; (100% pure)
1H NMR (400 MHz, MeOD-d4): δ ppm 8.85 (d, J = 4.9 Hz, 1H),
8.48 (s, 1H), 8.37−8.44 (m, 1H), 8.09 (s, 1H), 7.98 (d, J = 8.3 Hz,
1H), 7.87 (s, 1H), 7.60−7.83 (m, 1H), 7.18 (d, J = 8.8 Hz, 1H), 6.04
(d, J = 6.4 Hz, 1H), 4.25 (s, 3H), 3.91−4.04 (m, 1H), 3.40−3.55 (m,
4H), 1.90 (d, J = 6.4 Hz, 3H), 1.67−1.84 (m, 3H), 1.46−1.67 (m,
4H), 1.22−1.45 (m, 1H).
1.45 g, 11.32 mmol, 84% yield) as a colorless solid. H NMR (400
MHz, CDCl3-d): δ 3.97 (d, J = 8.8 Hz, 2H) 3.78 (d, J = 8.3 Hz, 2H)
2.28−2.20 (m, 2H) 1.64 (t, J = 3.4 Hz, 1H).
tert-Butyl (1R,5S,6r)-3-Oxabicyclo[3.1.0]hexan-6-ylcarbamate
(116). (1R,5S,6r)-3-Oxabicyclo[3.1.0]hexane-6-carboxylic acid (115,
1.45 g, 11.32 mmol) was suspended in toluene (20 mL), triethylamine
(5 mL, 35.9 mmol) and diphenyl phosphorazidate (3 mL, 13.95
mmol) were added, and the mixture was stirred for 20 min and then
tert-butanol (10 mL, 105 mmol) was added and the solution was
heated at reflux for 5 h. This was then diluted with EtOAc (50 mL)
and washed with water (50 mL) and saturated sodium bicarbonate
solution (50 mL). The organic layer was dried and evaporated in
vacuo to give a beige crystalline solid. The crude product was purified
by normal-phase chromatography on a 50 g silica column eluting with
0−100% EtOAc/cyclohexane to give tert-butyl (1R,5S,6r)-3-
oxabicyclo[3.1.0]hexan-6-ylcarbamate (116, 1.65 g, 8.28 mmol,
(S)-N-Methyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-
yl)ethoxy)benzamide (38). General procedure C was followed using
the following amounts: (S)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-
(pyridin-2-yl)ethoxy)benzoic acid (66, 0.311 g, 0.96 mmol), HATU
(0.365 g, 0.96 mmol), DIPEA (0.504 mL, 2.88 mmol), and DMF (5.6
mL). A 0.76 mL aliquot of this solution was added to methylamine
(0.120 mmol). The sample was purified by high pH MDAP, and the
solvent was subsequently dried under a stream of nitrogen to give (S)-
N-methyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)benzamide (38, 24.6 mg, 0.073 mmol, 55% yield). LC−MS
1
73.2% yield) as a colorless solid. H NMR (400 MHz, CDCl3-d): δ
4.65 (br s, 1H) 3.97 (d, J = 8.8 Hz, 2H) 3.72 (d, J = 8.3 Hz, 2H) 2.42
(d, J = 1.5 Hz, 1H) 1.78 (s, 2H) 1.46 (s, 9H).
(1R,5S,6r)-3-Oxabicyclo[3.1.0]hexan-6-amine, Hydrochloride
(117). tert-Butyl (1R,5S,6r)-3-oxabicyclo[3.1.0]hexan-6-ylcarbamate
(116, 1.65 g, 8.28 mmol) was dissolved in DCM (10 mL), and HCl
(10.35 mL, 41.4 mmol) was added. The mixture was stirred for 1 h at
rt and then evaporated in vacuo to give (1R,5S,6r)-3-oxabicyclo-
[3.1.0]hexan-6-amine, hydrochloride (117, 1.1 g, 8.11 mmol, 98%
yield) as a colorless solid. LC−MS (formic, ES+) tR = 0.62 min; m/z =
424.4; 1H NMR (400 MHz, DMSO-d6): δ 8.48 (br s, 2H) 3.81 (d, J =
8.8 Hz, 2H) 3.60 (d, J = 8.8 Hz, 2H) 2.24 (br t, J = 2.4 Hz, 1H) 2.08
(br t, J = 2.4 Hz, 2H).
N-((1R,5S,6r)-3-Oxabicyclo[3.1.0]hexan-6-yl)-3-(1-methyl-1H-
1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2yl)ethoxy)benzamide (40).
General procedure C was followed using the following amounts:
(S)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
benzoic acid (66, 50 mg, 0.154 mmol), HATU (70.3 mg, 0.185
mmol), DIPEA (0.081 mL, 0.462 mmol), DMF (5 mL), and
(1R,5S,6r)-3-oxabicyclo[3.1.0]hexan-6-amine hydrochloride (117,
22.99 mg, 0.170 mmol). The sample was purified by MDAP (high
pH method). The relevant fractions were combined and concentrated
in vacuo to give N-((1R,5S,6r)-3-oxabicyclo[3.1.0]hexan-6-yl)-3-(1-
methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)ethoxy)-
benzamide (40, 34 mg, 0.084 mmol, 54% yield) as an off-white solid.
LC−MS (high pH, ES+) tR = 0.79 min; m/z = 406.4; (100% pure);
1H NMR (400 MHz, MeOD-d4): δ 8.59 (d, J = 2.4 Hz, 1H) 8.57 (dt,
J = 4.4, 1.2 Hz, 1H) 8.48 (s, 1H), 7.78 (td, J = 7.5, 1.9 Hz, 1H) 7.62
(dd, J = 8.8, 2.0 Hz, 1H) 7.43 (d, J = 7.8 Hz, 1H) 7.33 (ddd, J = 7.3,
4.9, 1.5 Hz, 1H) 6.93 (d, J = 8.8 Hz, 1H), 5.68 (q, J = 6.4 Hz, 1H)
4.22 (s, 3H), 4.01 (d, J = 8.8 Hz, 2H) 3.74 (d, J = 8.3 Hz, 2H) 2.62 (t,
J = 2.4 Hz, 1H) 1.93 (br t, J = 2.9 Hz, 2H) 1.81 (d, J = 6.8 Hz, 3H).
(S)-3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
N-(tetrahydro-2H-pyran-4-yl)benzamide (41). General procedure C
was followed using the following amounts: (S)-3-(1-methyl-1H-1,2,3-
triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoic acid (66, 39 mg, 0.12
mmol), HATU (46 mg, 0.120 mmol), DIPEA (0.063 mL, 0.360
mmol), DMF (0.70 mL), and tetrahydro-2H-pyran-4-amine (0.120
mmol). The sample was purified by high pH MDAP. The solvent was
dried under a stream of nitrogen in the Radleys blowdown apparatus
to give (S)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)-N-(tetrahydro-2H-pyran-4-yl)benzamide (41, 22.6 mg,
0.055 mmol, 42% yield). LC−MS (formic, ES+) tR = 0.68 min; m/z
= 408.2; (100% pure) 1H NMR (600 MHz, DMSO-d6): δ 8.66−8.65
(m, 1H) 8.60−8.58 (m, 1H) 8.56 (s, 1H) 8.28−8.25 (m, 1H) 7.78−
7.75 (m, 1H) 7.67−7.64 (m, 1H) 7.40−7.37 (m, 1H) 7.32−7.29 (m,
1H) 7.00−6.98 (m, 1H) 5.75 (d, J = 6.8 Hz, 1H) 4.18 (s, 3H) 4.12−
4.08 (m, 1H) 3.87 (br d, J = 9.8 Hz, 2H) 3.3 (obs, m, 2H) 1.77−1.75
(m, 3H) 1.75−1.70 (m, 2H) 1.62−1.53 (m, 2H).
1
(formic, ES+) tR = 0.62 min; m/z = 338.3; (100% pure) H NMR
(600 MHz, DMSO-d6): δ 8.66 (d, J = 2.3 Hz, 1H) 8.60 (br d, J = 4.9
Hz, 1H) 8.55 (s, 1H) 8.35 (q, J = 4.5 Hz, 1H) 7.77 (td, J = 7.9, 1.5
Hz, 1H) 7.64 (dd, J = 8.7, 2.3 Hz, 1H) 7.40 (d, J = 7.9 Hz, 1H) 7.31
(ddd, J = 7.5, 4.9, 1.1 Hz, 1H) 6.99 (d, J = 8.7 Hz, 1H) 5.74 (q, J =
6.0 Hz, 1H) 4.18 (s, 3H) 2.76 (d, J = 4.5 Hz, 3H) 1.76 (d, J = 6.4 Hz,
3H).
(S)-N-Cyclopropyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyri-
din-2-yl)ethoxy)benzamide (39). General procedure C was followed
using the following amounts: HATU (41 mg, 0.108 mmol), (S)-3-(1-
methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoic acid
(66, 64 mg, 0.196 mmol), DMF (1.5 mL), DIPEA (0.075 mL,
0.431 mmol), and cyclopropanamine (23 mg, 0.403 mmol). This gave
(S)-N-cyclopropyl-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-
yl)ethoxy)benzamide (39, 27 mg, 0.074 mmol, 38% yield) as an
orange solid. LC−MS (formic, ES+) tR = 0.71 min; m/z = 364.2;
(100% pure) 1H NMR (400 MHz, MeOD-d4): δ 8.58 (d, J = 2.4 Hz,
1H) 8.57−8.56 (m, 1H) 8.49 (s, 1H) 8.37 (br s, 1H) 7.78 (td, J = 7.8,
2.0 Hz, 1H) 7.62 (dd, J = 8.3, 2.0 Hz, 1H) 7.46−7.41 (m, J = 7.8 Hz,
1H) 7.34 (ddd, J = 7.8, 4.9, 1.5 Hz, 1H) 6.93 (d, J = 8.8 Hz, 1H) 5.69
(q, J = 6.8 Hz, 1H) 4.22 (s, 3H) 2.88−2.80 (m, J = 3.9 Hz, 1H) 1.82
(d, J = 6.4 Hz, 3H) 0.83−0.76 (m, 2H), 0.66−0.60 (m, 2H).
Methyl 4-Hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoate
(114). To a mixture of methyl 4-(methoxymethoxy)-3-(1-methyl-
1H-1,2,3-triazol-4-yl)benzoate (57, 25.3760 g, 92 mmol) in methanol
(150 mL) in a 500 mL round-bottomed flask was added conc. HCl
(20 mL, 658 mmol). The reaction mixture was stirred at 50 °C for 20
h. The solvent was evaporated from the reaction mixture in vacuo, and
the residue was triturated in methanol (approx 150 mL) for 30 min.
The solid was filtered and washed with further methanol (approx 100
mL) and diethyl ether (approx 100 mL). The solid was dried in vacuo
to give methyl 4-hydroxy-3-(1-methyl-1H-1,2,3-triazol-4-yl)benzoate
(114, 17.8 g, 76 mmol, 83% yield) as a pale yellow solid. LC−MS
1
(formic, ES+) tR = 0.80 min; m/z = 234.3; H NMR (400 MHz,
DMSO-d6): δ 11.14−11.06 (m, 1H) 8.69 (d, J = 2.4 Hz, 1H) 8.48−
8.38 (m, 1H) 7.79 (dd, J = 8.3, 2.4 Hz, 1H) 7.07 (d, J = 8.8 Hz, 1H)
4.12 (s, 3H) 3.84 (s, 3H).
(1R,5S,6r)-3-Oxabicyclo[3.1.0]hexane-6-carboxylic Acid (115).
(1R,5S,6r)-Ethyl 3-oxabicyclo[3.1.0]hexane-6-carboxylate (2.1 g,
13.45 mmol) was dissolved in ethanol (20 mL), then NaOH (20
mL, 40.0 mmol) was added, and the mixture was stirred for 2 h at rt
and then evaporated to half volume in vacuo. The solution was washed
with ether (20 mL), then acidified with 2 M HCl to pH 4, and
extracted with DCM (4 × 20 mL) and 10% MeOH/DCM (2 × 20
mL). The combined organics were dried and evaporated in vacuo to
give (1R,5S,6r)-3-oxabicyclo[3.1.0]hexane-6-carboxylic acid (115,
(S)-3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
N-((tetrahydro-2H-pyran-4-yl)methyl)benzamide (42). General pro-
cedure C was followed using the following amounts: (S)-3-(1-methyl-
1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoic acid (66, 39
mg, 0.12 mmol), HATU (46 mg, 0.120 mmol), DIPEA (0.063 mL,
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0.360 mmol), DMF (0.70 mL), and (tetrahydro-2H-pyran-4-yl)-
methanamine (0.120 mmol). The sample was purified by high pH
MDAP. The solvent was dried under a stream of nitrogen in the
Radleys blowdown apparatus to give (S)-3-(1-methyl-1H-1,2,3-
triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-N-((tetrahydro-2H-pyran-4-
yl)methyl)benzamide (42, 19.6 mg, 0.047 mmol, 35% yield). LC−MS
diluted with MeOH and treated with 1 M NaOH (50 mL) and stirred
at rt for 2 h, a precipitate resulted. The reaction mixture was
concentrated to remove the MeOH and was diluted with water and
extracted with EtOAc, and the combined organics were washed with
water, dried using a hydrophobic frit, and concentrated to give a
colorless oil. This oil was further purified using silica chromatography
eluting with 0−8% 2 M NH3 in MeOH/DCM to give (R)-tert-butyl
2-(2-aminoethyl)morpholine-4-carboxylate (120, 965 mg, 4.19 mmol,
40% yield) as a colorless oil. 1H NMR (400 MHz, DMSO-d6): δ 3.73
(dd, J = 35.2, 11.7 Hz, 3H) 3.35 (td, J = 11.7, 3.4 Hz, 1H) 3.42−3.30
(m, 1H) 3.07−2.73 (m, 1H) 3.07−2.73 (m, 1H) 2.61 (td, J = 6.8, 2.9
Hz, 2H) 1.53−1.44 (m, 2H) 1.41 (s, 9H).
(R)-tert-Butyl 2-(2-(3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-
(pyridin-2-yl)ethoxy)benzamido)ethyl)morpholine-4-carboxylate
(60). General procedure C was followed using the following amounts:
(S)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
benzoic acid (66, 100 mg, 0.308 mmol), HATU (141 mg, 0.370
mmol), DIPEA (0.162 mL, 0.925 mmol), DMF (5 mL), and (R)-tert-
butyl 2-(2-aminoethyl)morpholine-4-carboxylate (120, 71.0 mg,
0.308 mmol). The sample was purified by silica chromatography
eluting with 0−25% 2 M NH3 in 20:80 MeOH/DCM. The relevant
fractions were combined and concentrated in vacuo to give (R)-tert-
butyl 2-(2-(3-(1-methyl-1H-1,2,3-triazol-4-yl)-4((S)-1-(pyridin-2-yl)-
ethoxy)benzamido)ethyl)morpholine-4-carboxylate (60, 146 mg,
0.272 mmol, 88% yield) as a yellow oil. LC−MS (formic, ES+) tR =
0.96 min; m/z = 537.2; 1H NMR (400 MHz, MeOD-d4): δ 8.61 (d, J
= 2.0 Hz, 1H) 8.58−8.56 (m, 1H) 8.49 (s, 1H) 8.32 (t, J = 5.4 Hz,
1H) 7.78 (td, J = 7.3, 1.5 Hz, 1H) 7.64 (dd, J = 8.3, 2.0 Hz, 1H) 7.43
(d, J = 7.8 Hz, 1H) 7.33 (ddd, J = 7.8, 5.4, 1.5 Hz, 1H) 6.94 (d, J =
8.8 Hz, 1H) 5.69 (q, J = 6.8 Hz, 1H) 4.22 (s, 3H) 3.92−3.86 (m, 2H)
3.83 (dt, J = 13.2, 1.0 Hz, 1H) 3.54−3.44 (m, 4H) 2.98−2.89 (m,
1H) 2.75−2.59 (m, 1H) 1.82 (d, J = 6.8 Hz, 3H) 1.80−1.72 (m, 2H)
1.45 (s, 9H).
3-(1-Methyl-1H-1,2,3-triazol-4-yl)-N-(2-((R)-morpholin-2-yl)-
ethyl)-4-((S)-1-(pyridin-2yl)ethoxy)benzamide (44). (R)-tert-Butyl 2-
(2-(3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)-
ethoxy)benzamido)ethyl)morpholine-4-carboxylate (60, 146 mg,
0.272 mmol) was dissolved in DCM (3 mL), and TFA (0.105 mL,
1.360 mmol) was added. The reaction mixture was stirred at rt for 2 h
after which TFA (0.4 mL, 5.19 mmol) was added. The reaction
mixture was stirred for 30 min. Sat. NaHCO3 solution (10 mL) was
added to quench the reaction, and the reaction mixture was left to
stand for 4 days. The reaction mixture was diluted with water (5 mL)
and extracted with DCM (3 × 30 mL). The organics were dried via a
hydrophobic frit and concentrated in vacuo. The residue was taken up
into DCM (3 mL) and purified by silica chromatography eluting with
0−25% 2 M NH3 in 20:80 MeOH/DCM. The relevant fractions were
combined and concentrated in vacuo to give the product 3-(1-methyl-
1H-1,2,3-triazol-4-yl)-N-(2-((R)-morpholin-2-yl)ethyl)-4-((S)-1-
(pyridin-2-yl)ethoxy)benzamide (44, 16.6 mg, 0.038 mmol, 14%
yield) as an off-white solid. LC−MS (formic, ES+) tR = 0.48 min; m/z
= 437.3; (100% pure) 1H NMR (400 MHz, MeOD-d4): δ 8.62−8.60
(m, 1H) 8.59−8.56 (m, 1H) 8.50 (s, 1H) 7.82−7.77 (m, 1H) 7.66−
7.62 (m, 1H) 7.46−7.42 (m, 1H) 7.37−7.32 (m, 1H) 6.95 (d, J = 8.8
Hz, 1H) 5.70 (d, J = 6.4 Hz, 1H) 4.23 (s, 3H) 3.95−3.90 (m, 1H)
3.70−3.56 (m, 2H) 3.48 (t, J = 6.8 Hz, 2H) 2.98−2.93 (m, 1H) 2.87
(s, 2H) 2.62 (d, J = 10.3 Hz, 1H) 1.83 (d, J = 6.8 Hz, 3H) 1.78−1.69
(m, 2H).
(R)-tert-Butyl 2-(((Methylsulfonyl)oxy)methyl)morpholine-4-car-
boxylate (121). (R)-tert-Butyl 2-(hydroxymethyl)morpholine-4-car-
boxylate (3 g, 13.81 mmol) and triethylamine (2.89 mL, 20.71 mmol)
were stirred in DCM (30 mL) at 0 °C, methanesulfonyl chloride
(1.184 mL, 15.19 mmol) was added portionwise over 5 min, and the
reaction mixture was stirred at rt for 1 h. The reaction mixture was
treated with further methanesulfonyl chloride (1.076 mL, 13.81
mmol) and triethylamine (1.925 mL, 13.81 mmol) and stirred at rt for
1 h. The reaction mixture was diluted with further DCM and was
washed with 1 M HCl (aq), NaHCO3 (aq), and water, dried using a
hydrophobic frit, and concentrated to give (R)-tert-butyl 2-
(((methylsulfonyl)oxy)methyl)morpholine-4-carboxylate (121, 4.402
1
(formic, ES+) tR = 0.71 min; m/z = 422.2; (100% pure) H NMR
(600 MHz, DMSO-d6): δ 8.66 (d, J = 1.9 Hz, 1H) 8.59 (br d, J = 4.9
Hz, 1H) 8.56 (s, 1H) 8.43 (t, J = 5.6 Hz, 1H) 7.77 (td, J = 7.9, 1.5 Hz,
1H) 7.66 (dd, J = 8.7, 2.3 Hz, 1H) 7.39 (d, J = 7.9 Hz, 1H) 7.31 (ddd,
J = 6.4, 4.9, 0.8 Hz, 1H) 6.99 (d, J = 9.0 Hz, 1H) 5.75 (q, J = 6.4 Hz,
1H) 4.18 (s, 3H) 3.83 (dd, J = 10.9, 2.6 Hz, 2H) 3.25 (t, J = 11.7 Hz,
2H) 3.13 (t, J = 6.4 Hz, 2H) 1.82−1.77 (m, J = 3.8 Hz, 1H) 1.76 (d, J
= 6.4 Hz, 3H) 1.57 (br d, J = 12.4 Hz, 2H) 1.18 (qd, J = 12.4, 4.5 Hz,
2H).
N-((trans)-4-Hydroxycyclohexyl)-3-(1-methyl-1H-1,2,3-triazol-4-
yl)-4-((S)-1-(pyridin-2-yl)ethoxy)benzamide (43). General procedure
C was followed using the following amounts: (S)-3-(1-methyl-1H-
1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)benzoic acid (66, 39 mg,
0.12 mmol), HATU (46 mg, 0.120 mmol), DIPEA (0.063 mL, 0.360
mmol), DMF (0.70 mL), and (trans)-4-aminocyclohexan-1-ol (14
mg, 0.120 mmol). The sample was purified by high pH MDAP. The
solvent was dried under a stream of nitrogen in the Radleys blowdown
apparatus to give N-((trans)-4-hydroxycyclohexyl)-3-(1-methyl-1H-
1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)ethoxy)benzamide (43, 18.5
mg, 0.044 mmol, 33% yield). LC−MS (formic, ES+) tR = 0.64 min;
1
m/z = 408.2; (100% pure) H NMR (600 MHz, DMSO-d6): δ ppm
8.63 (d, J = 2.3 Hz, 1H) 8.57−8.60 (m, 1H) 8.53−8.56 (m, 1H) 8.12
(d, J = 7.9 Hz, 1H) 7.76 (td, J = 7.6, 1.7 Hz, 1H) 7.63 (dd, J = 8.7, 2.3
Hz, 1H) 7.38 (d, J = 7.9 Hz, 1H) 7.30 (dd, J = 7.0, 5.5 Hz, 1H) 6.97
(d, J = 9.0 Hz, 1H) 5.71−5.77 (obs. q, 1H) 4.55 (br s, 1H) 4.18 (obs.
s, 3H) 4.06−4.14 (m, 1H) 3.64−3.75 (m, 1H) 1.84 (br d, J = 10.5
Hz, 2H) 1.72−1.81 (m, 4H) 1.31−1.42 (m, 2H) 1.19−1.28 (m, 2H).
(S)-tert-Butyl 2-(((Methylsulfonyl)oxy)methyl)morpholine-4-car-
boxylate (118). (S)-tert-Butyl 2-(hydroxymethyl)morpholine-4-car-
boxylate (3 g, 13.81 mmol) and triethylamine (3.85 mL, 27.6 mmol)
were stirred in DCM (30 mL) at 0 °C, methanesulfonyl chloride
(1.614 mL, 20.71 mmol) was added portionwise over 5 min, and the
reaction mixture was stirred at rt for 4 h. The reaction mixture was
diluted with further DCM and was washed with 1 N HCl (aq),
NaHCO3 (aq), and water, dried using a hydrophobic frit, and
concentrated to give (S)-tert-butyl 2-(((methylsulfonyl)oxy)methyl)-
morpholine-4-carboxylate (118, 4.242 g, 14.36 mmol, 104% yield) as
1
a yellow oil. H NMR (400 MHz, DMSO-d6): δ 4.29−4.17 (m, J =
25.9, 10.8, 3.4 Hz, 2H) 3.88−3.80 (m, J = 11.2 Hz, 2H) 3.71 (d, J =
13.2 Hz, 1H) 3.66−3.59 (m, J = 3.4, 2.0 Hz, 1H) 3.43 (td, J = 11.7,
2.9 Hz, 1H) 3.20 (s, 3H) 2.94−2.80 (m, 1H) 2.78−2.63 (m, 1H) 1.42
(s, 9H).
(R)-tert-Butyl 2-(Cyanomethyl)morpholine-4-carboxylate (119).
(S)-tert-Butyl 2-(((methylsulfonyl)oxy)methyl)morpholine-4-carbox-
ylate (118, 4.2 g, 14.22 mmol), KCN (0.972 g, 14.93 mmol), and KI
(3.54 g, 21.33 mmol) were stirred at 100 °C in DMSO (30 mL) for 4
h. The reaction mixture was diluted with water and was extracted with
EtOAc, the organic layer was washed with water and brine, dried
using a hydrophobic frit, and concentrated to a yellow oil. This oil was
purified using silica chromatography eluting with a gradient of 0−50%
EtOAc/cyclohexane to give (R)-tert-butyl 2-(cyanomethyl)-
morpholine-4-carboxylate (119, 2.393 g, 10.58 mmol, 74% yield) as
1
a white solid. H NMR (400 MHz, DMSO-d6): δ 3.89−3.82 (m, J =
11.7, 2.0 Hz, 2H) 3.70 (br d, J = 13.2 Hz, 1H) 3.63−3.55 (m, J = 2.9,
1.5 Hz, 1H) 3.45 (td, J = 11.2, 2.9 Hz, 1H) 2.85 (dd, J = 17.1, 4.4 Hz,
1H) 2.92−2.82 (m, 1H) 2.73 (dd, J = 17.1, 7.3 Hz, 1H) 2.77−2.60
(m, 1H) 1.41 (s, 9H).
(R)-tert-Butyl 2-(2-Aminoethyl)morpholine-4-carboxylate (120).
(R)-tert-Butyl 2-(cyanomethyl)morpholine-4-carboxylate (119, 2.39
g, 10.56 mmol) was taken up into THF (20 mL) and stirred at rt,
before the borane tetrahydrofuran complex (15.84 mL, 15.84 mmol)
was added over 10 min, and the reaction mixture was stirred at rt for 2
h. The reaction was quenched by the careful addition of MeOH until
all effervescence stopped. The reaction mixture was concentrated and
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g, 14.90 mmol, 108% yield) as a yellow oil. H NMR (400 MHz,
DMSO-d6): δ 4.29−4.17 (m, J = 10.8, 5.9, 3.4 Hz, 2H) 3.84 (d, J =
11.7 Hz, 2H) 3.70 (d, J = 13.2 Hz, 1H) 3.66−3.59 (m, 1H) 3.43 (td, J
= 11.7, 2.9 Hz, 1H) 3.20−3.19 (m, 3H) 2.93−2.82 (m, 1H) 2.76−
2.66 (m, 1H) 1.42 (s, 9H).
ethoxy)benzamido)ethyl)morpholine-4-carboxylate (61, 142 mg,
0.265 mmol) was dissolved in DCM (5 mL), and TFA (0.041 mL,
0.529 mmol) was added. The reaction mixture was stirred at rt for 2 h
after which TFA (0.5 mL, 6.49 mmol) and DCM (1 mL) were added.
The reaction mixture was stirred at rt for 1 h. The reaction mixture
was quenched with sat. NaHCO3 solution (10 mL), diluted with
water (5 mL), and extracted with DCM (3 × 30 mL). The organics
were dried using a hydrophobic frit and concentrated in vacuo to give
3-(1-methyl-1H-1,2,3-triazol-4-yl)-N-(2-((S)-morpholin-2-yl)ethyl)-
4-((S)-1-(pyridin-2-yl)ethoxy)benzamide (45, 87 mg, 0.199 mmol,
75% yield) as an off-white solid. LC−MS (formic, ES+) tR = 0.48 min;
(S)-tert-Butyl 2-(Cyanomethyl)morpholine-4-carboxylate (122).
(R)-tert-Butyl 2-(((methylsulfonyl)oxy)methyl)morpholine-4-carbox-
ylate (121, 4 g, 13.54 mmol), KCN (0.926 g, 14.22 mmol), and KI
(3.37 g, 20.31 mmol) were stirred at 80 °C in DMSO (30 mL) for 4 h
and then at 100 °C for 3 h. The reaction mixture was diluted with
water and was extracted with EtOAc. The organic layer was washed
with water and brine, dried using a hydrophobic frit, and concentrated
to a yellow oil. This oil was purified using silica chromatography,
eluting with a gradient of 0−50% EtOAc/cyclohexane to give (S)-tert-
butyl 2-(cyanomethyl)morpholine-4-carboxylate (122, 2.693 g, 11.90
1
m/z = 219.3; (100% pure) H NMR (400 MHz, MeOD-d4): δ 8.61
(d, J = 2.4 Hz, 1H) 8.58 (dd, J = 4.9, 1.0 Hz, 1H) 8.50−8.49 (m, 1H)
7.79 (tt, J = 7.8, 1.5 Hz, 1H) 7.64 (ddd, J = 8.8, 2.4, 1.0 Hz, 1H) 7.44
(d, J = 7.8 Hz, 1H) 7.36−7.32 (m, 1H), 6.95 (dd, J = 8.8, 1.0 Hz, 1H)
5.70 (q, J = 6.4 Hz, 1H) 4.23 (s, 3H) 3.88 (d, J = 11.2 Hz, 1H) 3.65−
3.52 (m, 2H) 3.47 (t, J = 6.8 Hz, 2H) 2.87 (d, J = 12.7 Hz, 1H) 2.81−
2.76 (m, 2H), 2.53 (dd, J = 13.2, 9.8 Hz, 1H) 1.83 (d, J = 6.4 Hz, 3H)
1.75−1.67 (m, 2H).
(R,E)-tert-Butyl 3-(3-Ethoxy-3-oxoprop-1-en-1-yl)-3-fluoropiperi-
dine-1-carboxylate (124). (S)-tert-Butyl 3-fluoro-3-(hydroxymethyl)-
piperidine-1-carboxylate (10 g, 42.9 mmol) was dissolved in DCM
(60 mL), and Dess−Martin periodinane (23.64 g, 55.7 mmol) was
added. The mixture was stirred at rt for 18 h and then washed with
water, and the organic layer was dried over sodium sulphate and
1
mmol, 88% yield) as a white solid. H NMR (400 MHz, DMSO-d6):
δ 3.90−3.81 (m, J = 11.7 Hz, 2H) 3.70 (d, J = 13.7 Hz, 1H) 3.63−
3.55 (m, 1H) 3.45 (td, J = 11.7, 2.9 Hz, 1H) 2.85 (dd, J = 17.1, 4.4
Hz, 1H) 2.92−2.81 (m, 1H) 2.73 (dd, J = 17.6, 7.8 Hz, 1H) 2.77−
2.57 (m, 1H) 1.42 (s, 9H).
(S)-tert-Butyl 2-(2-Aminoethyl)morpholine-4-carboxylate (123).
(S)-tert-Butyl 2-(cyanomethyl)morpholine-4-carboxylate (122, 2.6 g,
11.49 mmol) was taken up into THF (20 mL) and stirred at rt. The
borane tetrahydrofuran complex (17.24 mL, 17.24 mmol) was added
over 10 min, and the reaction mixture was stirred at rt for 2 h. The
reaction was quenched by the careful addition of MeOH until all
effervescence stopped. The reaction mixture was concentrated and
diluted with MeOH and treated with 1 M NaOH (50 mL) and stirred
at rt for 2 h, a precipitate resulted. The reaction mixture was
concentrated to remove the MeOH and was diluted with water and
extracted with EtOAc. The combined organics were washed with
water, dried using a hydrophobic frit, and concentrated to a colorless
oil. The oil was again taken up into THF (20 mL), treated with the
borane tetrahydrofuran complex (17.24 mL, 17.24 mmol), and stirred
at 70 °C under reflux conditions for 4 h. The reaction was quenched
by the careful addition of MeOH until all effervescence stopped. The
reaction mixure was concentrated and diluted with MeOH and
treated with 1 M NaOH (50 mL) and stirred at rt for 1 h, a
precipitate resulted. The reaction mixture was concentrated to remove
the MeOH and was diluted with water and extracted with EtOAc. The
combined organics were washed with water, dried using a hydro-
phobic frit, and concentrated to give (S)-tert-butyl 2-(2-aminoethyl)-
morpholine-4-carboxylate (122, 763 mg, 3.31 mmol, 29% yield) as a
d e c a n t e d i n t o
a
c l e a n , d r y fl a s k . E t h y l 2 -
(triphenylphosphoranylidene)acetate (19.41 g, 55.7 mmol) was
added, and the mixture was stirred overnight and then washed with
water, and the organic layer was dried and evaporated in vacuo. The
residue was purified by silica chromatography eluting with 0−50%
EtOAc/cyclohexane, and product-containing fractions were evapo-
rated in vacuo to give (R,E)-tert-butyl-3-(3-ethoxy-3-oxoprop-1-en-1-
yl)-3-fluoropiperidine-1-carboxylate (124, 10.5 g, 34.8 mmol, 81%
yield) as a colorless oil. 1H NMR (400 MHz, CDCl3-d): δ 6.90 (dd, J
= 19.6, 15.7 Hz, 1H) 6.16 (d, J = 15.6 Hz, 1H) 4.23 (q, J = 6.8 Hz,
2H) 4.07−3.96 (m, 1H) 3.92 (d, J = 13.2 Hz, 1H) 3.24−3.03 (m,
1H) 3.01−2.92 (m, J = 11.7 Hz, 1H) 2.02−1.92 (m, 1H) 1.89−1.79
(m, 1H) 1.77−1.65 (m, 1H) 1.63−1.54 (m, 1H) 1.47 (s, 9H) 1.32 (t,
J = 6.8 Hz, 3H).
(R)-tert-Butyl 3-(3-Ethoxy-3-oxopropyl)-3-fluoropiperidine-1-car-
boxylate (125). (R,E)-tert-Butyl 3-(3-ethoxy-3-oxoprop-1-en-1-yl)-3-
fluoropiperidine-1-carboxylate (124, 10 g, 33.2 mmol) was dissolved
in ethanol (100 mL) and added to Pd-C 5% (2 g, 18.79 mmol) under
N2. The mixture was then hydrogenated at atmospheric pressure for 6
h. The mixture was filtered through celite under nitrogen, and the
filtrate was evaporated in vacuo to give (R)-tert-butyl 3-(3-ethoxy-3-
oxopropyl)-3-fluoropiperidine-1-carboxylate (125, 9.5 g, 31.3 mmol,
94% yield) as a pale yellow oil. 1H NMR (400 MHz, CDCl3-d): δ 4.15
(q, J = 7.3 Hz, 2H) 3.96−3.82 (m, 1H) 3.76 (dt, J = 13.7, 4.4 Hz, 1H)
3.19−2.94 (m, 1H) 3.03 (br t, J = 9.8 Hz, 1H) 2.48 (t, J = 7.8 Hz,
2H) 2.01−1.87 (m, 3H) 1.85−1.73 (m, 1H) 1.64−1.50 (m, 2H) 1.47
(s, 9H) 1.27 (t, J = 6.8 Hz, 3H).
(R)-tert-Butyl 3-Fluoro-3-(3-hydroxypropyl)piperidine-1-carboxy-
late (126). LiBH4 (2.046 g, 94 mmol) was added to a solution of (R)-
tert-butyl 3-(3-ethoxy-3-oxopropyl)-3-fluoropiperidine-1-carboxylate
(125, 9.5 g, 31.3 mmol) in THF (100 mL), and the mixture was
stirred at rt under nitrogen for 48 h. The mixture was then cooled in
an ice bath and quenched very cautiously, initially dropwise addition
of ammonium chloride solution (100 mL). The mixture was then
stirred for 20 min and diluted with EtOAc (100 mL), and the
combined organics were separated, dried over sodium sulphate, and
evaporated in vacuo. The crude material was dissolved in DCM and
purified by silica chromatography eluting with 0−100% EtOAc/
cyclohexane, and product-containing fractions were evaporated in
vacuo to give (R)-tert-butyl 3-fluoro-3-(3-hydroxypropyl)piperidine-1-
carboxylate (126, 6.0 g, 23.0 mmol, 73% yield). 1H NMR (400 MHz,
CDCl3-d): δ 3.91−3.74 (m, 2H) 3.73−3.64 (m, 2H) 3.14−2.96 (m, J
= 14.2 Hz, 2H) 2.00−1.90 (m, 1H) 1.86−1.58 (m, 7H) 1.47 (s, 9H).
(R)-tert-Butyl 3-Fluoro-3-(3-((methylsulfonyl)oxy)propyl)-
piperidine-1-carboxylate (127). (R)-tert-Butyl 3-fluoro-3-(3-
1
colorless viscous oil. H NMR (400 MHz, DMSO-d6): δ 3.80−3.65
(m, J = 34.2, 11.7 Hz, 3H) 3.35 (td, J = 11.7, 2.9 Hz, 1H) 3.41−3.27
(m, 1H) 3.41−3.27 (m, 1H) 2.89−2.79 (m, 1H) 2.62−2.57 (m, 2H)
1.52−1.42 (m, 2H) 1.41 (s, 9H).
(S)-tert-Butyl 2-(2-(3-(1-Methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-
(pyridin-2-yl)ethoxy)benzamido)ethyl)morpholine-4-carboxylate
(61). General procedure C was followed using the following amounts:
(S)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)ethoxy)-
benzoic acid (66, 100 mg, 0.308 mmol), DIPEA (0.162 mL, 0.925
mmol), HATU (141 mg, 0.370 mmol), DMF (5.5 mL), and (S)-tert-
butyl 2-(2-aminoethyl)morpholine-4-carboxylate (123, 78 mg, 0.339
mmol). The sample was purified by silica chromatography eluting
with 0−5% MeOH/DCM. The relevant fractions were combined and
concentrated in vacuo to give (S)-tert-butyl 2-(2-(3-(1-methyl-1H-
1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)ethoxy)benzamido)ethyl)-
morpholine-4-carboxylate (61, 142 mg, 0.265 mmol, 86% yield), a
colorless oil. LC−MS (formic, ES+) tR = 0.96 min; m/z = 537.2; H
1
NMR (400 MHz, MeOD-d4): δ 8.61 (d, J = 2.4 Hz, 1H) 8.57 (dt, J =
4.9, 1.0 Hz, 1H) 8.50−8.49 (m, 1H) 7.79 (d, J = 2.0 Hz, 1H) 7.64
(dd, J = 8.3, 2.0 Hz, 1H) 7.44 (dt, J = 7.8, 1.0 Hz, 1H) 7.34 (ddd, J =
7.3, 4.9, 1.0 Hz, 1H) 6.95 (d, J = 8.8 Hz, 1H) 5.69 (q, J = 6.8 Hz, 1H)
4.23 (s, 3H) 3.93−3.86 (m, 2H) 3.83 (dt, J = 13.7, 1.0 Hz, 1H) 3.52−
3.47 (m, 4H) 2.99−2.90 (m, 1H) 2.74−2.63 (m, 1H) 1.82 (d, J = 6.8
Hz, 3H) 1.80−1.72 (m, 2H) 1.45 (s, 9H).
3-(1-Methyl-1H-1,2,3-triazol-4-yl)-N-(2-((S)-morpholin-2-yl)-
ethyl)-4-((S)-1-(pyridin-2-yl)ethoxy)benzamide (45). (S)-tert-Butyl
2-(2-(3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)-
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hydroxypropyl)piperidine-1-carboxylate (126, 6 g, 22.96 mmol) was
dissolved in DCM (100 mL). Et3N (4.80 mL, 34.4 mmol) was added,
and the mixture was cooled in an ice bath, then methanesulfonyl
chloride (2.326 mL, 29.8 mmol) was added dropwise, and the mixture
was stirred for 2 h, allowing it to warm to rt. The solution was washed
with water (100 mL) and brine (100 mL), and the organic layer was
dried and evaporated in vacuo to give (R)-tert-butyl 3-fluoro-3-
(3((methylsulfonyl)oxy)propyl)piperidine-1-carboxylate (127, 7.2 g,
21.21 mmol, 92% yield) as a colorless oil. 1H NMR (400 MHz,
CDCl3-d): δ 4.28 (td, J = 6.4, 2.9 Hz, 2H) 3.91−3.72 (m, 2H) 3.02 (s,
3H) 3.15−2.99 (m, 2H) 1.95 (quin, J = 7.3 Hz, 3H) 1.84−1.52 (m,
5H) 1.48 (s, 9H).
idin-2-yl)ethoxy)benzamido)propyl)piperidine-1-carboxylate (62,
160 mg, 0.282 mmol) was dissolved in DCM (5 mL), and TFA
(0.5 mL, 6.49 mmol) was added. The reaction mixture was stirred at
rt for 1 h. Saturated NaHCO3 solution (10 mL) was added, and the
mixture was stirred for 1 h. The reaction mixture was diluted with
water (5 mL) and extracted with DCM (3 × 30 mL). Brine (5 mL)
was added. The organics were washed with 10% LiCl solution (equal
volume), dried via a hydrophobic frit, and concentrated in vacuo. The
residue was taken up into MeOH (2 mL) and eluted through an NH2
Isolute column (prewashed with MeOH) with MeOH (50 mL). The
relevant fractions were combined and concentrated in vacuo. The
residue was taken up into 3:1 MeOH/DMSO (1 mL) and purified by
MDAP (high pH method). The relevant fractions were combined and
concentrated in vacuo to give the product N-(3-((R)-3-fluoropiper-
idin-3-yl)propyl)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-(pyri-
din-2-yl)ethoxy)benzamide (46, 24 mg, 0.051 mmol, 18% yield) as an
off-white solid. LC−MS (high pH, ES+) tR = 0.85 min; m/z = 467.3;
(100% pure) 1H NMR (400 MHz, MeOD-d4): δ 8.62 (d, J = 2.4 Hz,
1H) 8.57 (ddd, J = 4.9, 1.0, 1.0 Hz, 1H) 8.49 (s, 1H) 7.78 (td, J = 7.8,
1.5 Hz, 1H) 7.64 (dd, J = 8.8, 2.4 Hz, 1H) 7.45−7.42 (m, J = 8.3 Hz,
1H) 7.33 (ddd, J = 7.3, 4.9, 1.0 Hz, 1H) 6.94 (d, J = 8.8 Hz, 1H) 5.68
(q, J = 6.8 Hz, 1H) 4.22 (s, 3H) 3.37 (t, J = 6.8 Hz, 2H) 3.00−2.90
(m, J = 12.2, 2.9 Hz, 2H) 2.69−2.50 (m, 2H) 1.99−1.90 (m, 1H)
1.82 (d, J = 6.4 Hz, 3H) 1.76−1.51 (m, 7H).
tert-Butyl 2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (73). tert-Butyl 2-chloro-6-(1-methyl-1H-1,2,3-triazol-
4-yl)isonicotinate (69, 400 mg, 1.357 mmol), PdCl2(PPh3)2 (95 mg,
0.136 mmol), and benzylzinc(II) bromide (4.07 mL, 2.036 mmol)
were dissolved in THF (6 mL). The reaction mixture was then
irradiated for 0.5 h at 100 °C. The reaction mixture was partitioned
between EtOAc (25 mL) and water (30 mL). The aqueous layer was
removed; the organic layer was washed (1× water 20 mL, 2× sat. aq
NaHCO3 20 mL), passed through a hydrophobic frit, and evaporated
in vacuo to a red/brown oil. The sample was then dissolved in 5 mL of
DCM and was purified by silica chromatography eluting with 0−50%
EtOAc/cyclohexane. The product-containing fractions were com-
bined, and the solvent was removed in vacuo. The sample was then
dried under a stream of nitrogen for 16 h and was placed in a vacuum
oven for 30 min to give tert-butyl 2-benzyl-6-(1-methyl-1H-1,2,3-
triazol-4-yl)isonicotinate (73, 261 mg, 0.559 mmol, 41% yield) as a
red/brown gum. LC−MS (formic, ES+) tR = 1.25 min; m/z = 351.3;
1H NMR (400 MHz, DMSO-d6): δ ppm 8.62 (s, 1H) 8.21 (d, J = 1.5
(R)-tert-Butyl 3-(3-Azidopropyl)-3-fluoropiperidine-1-carboxy-
late (128). Sodium azide (2.68 g, 41.2 mmol) was added to a
solution of (R)-tert-butyl 3-fluoro-3-(3-((methylsulfonyl)oxy)propyl)-
piperidine-1-carboxylate (127, 7 g, 20.62 mmol) in DMF (50 mL),
and the mixture was heated at 70 °C for 2 h and then diluted with
water (200 mL) and extracted with EtOAc (2 × 100 mL). The
combined organics were washed with water (2 × 100 mL), dried, and
evaporated in vacuo. The crude product was dissolved in DCM (10
mL) and purified by silica chromatography eluting with 0−50%
EtOAc/cyclohexane, and product-containing fractions (visualized by
ninhydrin) were evaporated in vacuo to give (R)-tert-butyl 3-(3-
azidopropyl)-3-fluoropiperidine-1-carboxylate (128, 5.2 g, 18.16
1
mmol, 88% yield) as a colorless oil. H NMR (400 MHz, CDCl3-
d): δ 3.77 (dt, J = 13.2, 4.4 Hz, 1H) 3.98−3.72 (m, 1H) 3.34 (t, J =
6.6 Hz, 2H) 3.16−2.99 (m, 1H) 3.16−2.99 (m, J = 12.7, 10.3 Hz,
1H) 1.99−1.89 (m, 1H) 1.84−1.54 (m, 7H) 1.48 (s, 9H).
(S)-tert-Butyl 3-(3-Aminopropyl)-3-fluoropiperidine-1-carboxy-
late (129). (R)-tert-Butyl 3-(3-azidopropyl)-3-fluoropiperidine-1-
carboxylate (128, 5.0 g, 17.46 mmol) was dissolved in THF (50
mL), and triphenylphosphine (5.50 g, 20.95 mmol) was added. The
mixture was then stirred at rt over the weekend. Water (50 mL) was
added, and the mixture was stirred vigorously for 2 h and then diluted
with EtOAc (100 mL) and brine (50 mL). The organic layer was
separated, dried, and evaporated in vacuo. The crude product was
dissolved in DCM (20 mL) and purified by silica chromatography
eluting with 0−20% 2 M methanolic ammonia/DCM. Product-
containing fractions were evaporated in vacuo to give (S)-tert-butyl 3-
(3-aminopropyl)-3-fluoropiperidine-1-carboxylate (129, 4.0 g, 15.36
mmol, 88% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3-d): δ
3.99−3.74 (m, 1H) 3.81 (br d, J = 10.8 Hz, 1H) 3.11−2.92 (m, 1H)
3.11−2.92 (m, J = 11.7, 10.3 Hz, 1H) 2.76−2.70 (m, J = 6.8 Hz, 2H)
1.98−1.88 (m, 1H) 1.86−1.74 (m, 1H) 1.71−1.50 (m, 6H) 1.47 (s,
9H) 1.39 (br s, 2H).
Hz, 1H) 7.59 (d, J = 1.5 Hz, 1H) 7.28−7.45 (m, 4H) 7.19−7.26 (m,
1H) 4.22 (obs. s, 2H) 4.13 (s, 3H) 1.57 (s, 9H).
2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinamide (47).
2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic acid (82, 30
mg, 0.102 mmol), EDC (23.45 mg, 0.122 mmol), 1H-benzo[d]-
[1,2,3]triazol-1-ol, ammonia salt (31.0 mg, 0.204 mmol), and DIPEA
(0.053 mL, 0.306 mmol) were dissolved in DMF (4 mL). The
reaction mixture was partitioned between EtOAc (25 mL) and water
(30 mL). The organic layer was washed (1× water 20 mL, 2× sat. aq
NaHCO3 20 mL), passed through a hydrophobic frit, and evaporated
in vacuo to a white solid. The sample was then purified by silica
chromatography 20−100% EtOAc/cyclohexane. The product-con-
taining fractions were combined, and the solvent was removed in
vacuo. The sample was then dried under a stream of nitrogen for 1 h
and was placed in the vacuum oven for 30 min to give 2-benzyl-6-(1-
methyl-1H-1,2,3-triazol-4-yl)isonicotinamide (47, 14 mg, 0.048
mmol, 47% yield) as a white solid. LC−MS (formic, ES+) tR = 0.77
(S)-tert-Butyl 3-Fluoro-3-(3-(3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-
((S)-1-(pyridin-2-yl)ethoxy)benzamido)propyl)piperidine-1-carbox-
ylate (62). General procedure C was followed using the following
amounts: (S)-3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-(1-(pyridin-2-yl)-
ethoxy)benzoic acid (66, 100 mg, 0.308 mmol), HATU (141 mg,
0.370 mmol), DIPEA (0.162 mL, 0.925 mmol), DMF (5 mL), and
(S)-tert-butyl 3-(3-aminopropyl)-3-fluoropiperidine-1-carboxylate
(129, 88 mg, 0.339 mmol). The sample was purified by silica
chromatography eluting with 0−25% 2 M NH3 in 20:80 MeOH/
DCM in DCM. The relevant fractions were combined and
concentrated in vacuo to give the product (S)-tert-butyl 3-fluoro-3-
(3-(3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-(pyridin-2-yl)-
ethoxy)benzamido)propyl)piperidine-1-carboxylate (62, 160 mg,
0.282 mmol, 92% yield) as a yellow/orange oil. LC−MS (high pH,
ES+) tR = 1.12 min; m/z = 567.5; 1H NMR (400 MHz, MeOD-d4): δ
8.63 (d, J = 2.4 Hz, 1H) 8.56 (dt, J = 4.4, 1.5 Hz, 1H) 8.48 (s, 1H)
7.77 (td, J = 7.8, 1.5 Hz, 1H) 7.64 (dd, J = 8.8, 2.4 Hz, 1H) 7.43 (d, J
= 8.3 Hz, 1H) 7.32 (ddd, J = 7.3, 4.9, 1.0 Hz, 1H) 6.93 (d, J = 8.8 Hz,
1H) 5.67 (q, J = 6.4 Hz, 1H) 4.21 (s, 3H) 3.86 (br dt, J = 13.2, 3.9
Hz, 1H) 3.93 (br t, J = 11.7 Hz, 1H) 3.38 (t, J = 6.8 Hz, 2H) 3.12−
2.81 (m, 2H) 1.96−1.87 (m, 1H) 1.81 (d, J = 6.4 Hz, 3H) 1.76−1.51
(m, 7H) 1.43 (s, 9H).
1
min; m/z = 294.2; (98% pure) H NMR (400 MHz, DMSO-d6): δ
ppm 8.59 (s, 1H) 8.35 (br s, 1H) 8.27 (d, J = 1.5 Hz, 1H) 7.71 (br s,
1H) 7.60 (d, J = 1.5 Hz, 1H) 7.27−7.38 (m, 4H) 7.18−7.25 (m, 1H)
4.17 (s, 2H) 4.13 (s, 3H).
Methyl 2-Benzyl-6-bromoisonicotinate (130). A yellow solution
of methyl 2,6-dibromopyridine-4-carboxylate (2.5 g, 8.48 mmol) and
tetrakis triphenyl phosphine palladium (0.40 g, 0.346 mmol) was
sparged with nitrogen for 15 min and a solution of 0.5 M
benzylzinc(II) bromide in THF (20 mL, 10.00 mmol) was added.
The resulting brown solution was heated to 60 °C under nitrogen for
20 min to give a black solution. MeOH (5 mL) was added to quench
N-(3-((R)-3-Fluoropiperidin-3-yl)propyl)-3-(1-methyl-1H-1,2,3-tri-
azol-4-yl)-4-((S)-1-(pyridin-2-yl)ethoxy)benzamide (46). (S)-tert-
Butyl 3-fluoro-3-(3-(3-(1-methyl-1H-1,2,3-triazol-4-yl)-4-((S)-1-(pyr-
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the reaction, and the mixture was cooled to rt. The reaction mixture
was stood overnight and diluted with EtOAc, and the solution was
washed twice with sat. aq NH4Cl. The organic layer was dried over
MgSO4 and evaporated in vacuo to a yellow oil. Cyclohexane was
added to the residue and a suspension formed. The suspension was
stirred vigorously for 30 min and filtered. The filtrate was loaded onto
a 100 g Biotage silica SNAP column and eluted with 0−10% EtOAc/
cyclohexane. The product-containing fractions were evaporated in
vacuo to give methyl 2-benzyl-6-bromoisonicotinate (130, 1.837 g,
6.00 mmol, 64% yield) as a yellow oil. LC−MS (TFA, ES+) tR = 1.24
min; m/z = 306.0, 308.0; 1H NMR (400 MHz, DMSO-d6): δ 7.82 (d,
J = 1.5 Hz, 1H) 7.75 (d, J = 1.5 Hz, 1H) 7.36−7.27 (m, 5H) 4.20−
4.18 (m, 2H) 3.88 (s, 3H).
Methyl 2-Benzyl-6-((trimethylsilyl)ethynyl)isonicotinate (131).
Tetrakis triphenylphosphine palladium (0.122 g, 0.106 mmol) was
added to a rapidly stirred yellow solution of methyl 2-benzyl-6-
bromoisonicotinate (130, 1.62 g, 5.29 mmol), (trimethylsilyl)-
acetylene (4.52 mL, 32.0 mmol), copper(I) iodide (0.040 g, 0.212
mmol), and diisopropylamine (4.52 mL, 31.7 mmol). The suspension
was stirred for 3 h. The reaction mixture was filtered, and the
remaining solid was washed with EtOAc. The filtrate was washed with
water (×2) and brine, and an emulsion formed. The emulsion was
passed through a phase separator as were the combined aqueous
washings. The aqueous portion was extracted with DCM, and the
combined organics were evaporated in vacuo to a black gum. The
residue was dissolved in toluene and purified by silica chromatography
eluting with 10−50% DCM/hexane. The product-containing fractions
were evaporated in vacuo to give methyl 2-benzyl-6-((trimethylsilyl)-
ethynyl)isonicotinate (131, 1756 mg, 4.89 mmol, 92% yield) as a
black oil. LC−MS (TFA, ES+) tR = 1.46 min; m/z = 324.2; 1H NMR
(400 MHz, DMSO-d6): δ 7.71 (s, 2H) 7.35−7.20 (m, 5H) 4.19 (s,
2H) 3.87 (s, 3H) 0.27 (s, 9H).
4.13 (s, 3H) 3.87 (d, J = 8.5 Hz, 1H) 3.64 (br d, J = 8.2 Hz, 2H) 2.64
(dt, J = 4.4, 2.5 Hz, 1H) 1.94 (s, 2H).
2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-N-(tetrahydro-2H-
pyran-4-yl)isonicotinamide (49). General procedure C was followed
using the following amounts: 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-
yl)isonicotinic acid (82, 0.029 g, 0.1 mmol), HATU (0.038 g, 0.100
mmol), DMF (0.5 mL), DIPEA (0.055 mL, 0.315 mmol), and
tetrahydro-2H-pyran-4-amine (0.120 mmol). The sample was purified
by high pH MDAP. The solvent was dried under a stream of nitrogen
in the Radleys blowdown apparatus to give 2-benzyl-6-(1-methyl-1H-
1,2,3-triazol-4-yl)-N-(tetrahydro-2H-pyran-4-yl)isonicotinamide (49,
8.2 mg, 0.022 mmol, 20% yield). LC−MS (formic, ES+) tR = 0.89
1
min; m/z = 378.2; (100% pure) H NMR (600 MHz, DMSO-d6): δ
8.75 (br d, J = 7.9 Hz, 1H) 8.59 (s, 1H) 8.29−8.25 (m, J = 1.1 Hz,
1H) 7.60−7.56 (m, J = 1.1 Hz, 1H) 7.35−7.30 (m, 3H) 7.22 (t, J =
7.5 Hz, 1H) 4.18 (s, 2H) 4.13 (s, 3H) 4.06−3.98 (m, 1H) 3.88 (d, J =
11.7 Hz, 2H) 3.39 (t, J = 11.7 Hz, 2H) 1.77 (d, J = 12.8 Hz, 2H) 1.60
(qd, J = 11.7, 4.5 Hz, 2H).
2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-N-((tetrahydro-2H-
pyran-4-yl)methyl)isonicotinamide (50). General procedure C was
followed using the following amounts: HATU (78 mg, 0.204 mmol),
2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic acid (82, 50
mg, 0.170 mmol), (tetrahydro-2H-pyran-4-yl)methanamine (0.031
mL, 0.255 mmol), DIPEA (0.089 mL, 0.510 mmol), and DMF (4
mL). The sample was purified using normal-phase column
chromatography, eluting with a gradient of 80−100% EtOAc/
cyclohexane. The product-containing fractions were combined, and
the solvent was removed in vacuo. The sample was then dried under a
stream of nitrogen for 1 h and was then placed in the vacuum oven at
40 °C 1 h to give 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-N-
((tetrahydro-2H-pyran-4-yl)methyl)isonicotinamide (50, 50 mg, 0.12
mmol, 68% yield) as a white solid. LC−MS (formic, ES+) tR = 0.91
1
min; m/z = 392.3; (100% pure) H NMR (400 MHz, DMSO-d6): δ
2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate, Sodium
Salt (132). Methyl 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinate (131, 955 mg, 3.10 mmol) was suspended in
isopropanol (50 mL) and heated to reflux. The resulting solution
was removed from the heat and a solution of 2 M NaOH (3.10 mL,
6.19 mmol) was added. The mixture was cooled to rt with vigorous
stirring over 30 min. A white precipitate formed and the suspension
was stopped stirring. TBME (40 mL) was added; the suspension was
mechanically broken up and stirred vigorously for 20 min. The
mixture was then filtered; the solid was washed with TBME (3 × 10
mL) and dried in vacuo to give 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-
4-yl)isonicotinate, sodium salt (132, 994 mg, 2.83 mmol, 91% yield)
as a white solid. LC−MS (TFA, ES+) tR = 0.84 min; m/z = 295.2; 1H
NMR (400 MHz, DMSO-d6): δ ppm 8.19 (d, J = 1.5 Hz, 1H) 7.45
(d, J = 1.5 Hz, 1H) 7.26−7.35 (m, 5H) 7.16−7.24 (m, 1H) 4.04−
4.15 (m, 5H).
2-Benzyl-N-((1R,5S,6r)-3-oxabicyclo[3.1.0]hexan-6-yl)-6-(1-meth-
yl-1H-1,2,3-triazol-4-yl)isonicotinamide (48). A solution of 2-benzyl-
6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinate, sodium salt (132,
0.032 g, 0.1 mmol) and HATU (0.038 g, 0.100 mmol) dissolved in
DMF (0.5 mL) was prepared. DIPEA (0.055 mL, 0.315 mmol) was
added, and the mixture was shaken and added to preweighed
(1R,5S,6r)-3-oxabicyclo[3.1.0]hexan-6-amine (117, 0.120 mmol).
The reaction mixture was capped, shaken, and left to stand at rt for
2 h. HATU (20 mg) was added to the reaction mixture with DIPEA
(30 μL). The mixture was capped, shaken, and left to stand at rt for
18 h. A solution of T3P 50% in EtOAC (120 μL) and DIPEA (55 μL)
was added to the reaction vial. The reaction mixture was capped,
shaken, and stood at rt for 1 h. The solvent was removed from the
reaction mixture. The sample was dissolved in DMSO (2 mL) and
purified by high pH MDAP. The solvent was dried under a stream of
nitrogen in the Radleys blowdown apparatus to give 2-benzyl-N-
((1R,5S,6r)-3-oxabicyclo[3.1.0]hexan-6-yl)-6-(1-methyl-1H-1,2,3-tria-
zol-4-yl)isonicotinamide (48, 9.6 mg, 0.026 mmol, 23% yield). LC−
MS (formic, ES+) tR = 0.88 min; m/z = 376.0; (100% pure) 1H NMR
(500 MHz, DMSO-d6): δ 8.24 (d, J = 1.4 Hz, 1H) 7.55 (d, J = 1.4 Hz,
1H) 7.35−7.30 (m, 4H) 7.22 (tt, J = 6.9, 1.9 Hz, 2H) 4.18 (s, 2H)
8.87 (t, J = 5.9 Hz, 1H) 8.58 (s, 1H) 8.26 (d, J = 1.5 Hz, 1H) 7.57 (d,
J = 1.5 Hz, 1H) 7.36−7.29 (m, 4H) 7.22 (tt, J = 6.8, 1.5 Hz, 1H) 4.18
(s, 2H) 4.13 (s, 3H) 3.85 (ddd, J = 11.7, 3.9, 2.0 Hz, 2H) 3.25 (dd, J
= 11.7, 2.0 Hz, 2H) 3.17 (t, J = 6.4 Hz, 2H) 1.86−1.76 (m, J = 3.9,
Hz, 1H) 1.60 (dd, J = 12.7, 2.0 Hz, 2H) 1.20 (qd, J = 12.2, 3.9 Hz,
2H).
(R)-tert-Butyl 2-(2-(2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamido)ethyl)morpholine-4-carboxylate (75). General pro-
cedure C was followed using the following amounts: DIPEA (0.095
mL, 0.544 mmol), 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinic acid (82, 100 mg, 0.272 mmol), (R)-tert-butyl 2-(2-
aminoethyl)morpholine-4-carboxylate (68.9 mg, 0.299 mmol),
HATU (134 mg, 0.353 mmol), and DMF (3 mL). The sample was
purified by silica chromatography using a gradient of 20−90%
EtOAc/cyclohexane. The product-containing fractions were com-
bined, and the solvent was removed under reduced pressure. The
product was left to dry in vacuo for 2 h to give (R)-tert-butyl 2-(2-(2-
benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinamido)ethyl)-
morpholine-4-carboxylate (75, 104 mg, 0.164 mmol, 60% yield) as a
1
yellow gum. LC−MS (formic, ES+) tR = 1.12 min; m/z = 507.4; H
NMR (400 MHz, CDCl3-d): δ 8.19 (d, J = 1.5 Hz, 1H) 8.18 (s, 1H)
7.73−7.66 (m, 1H) 7.55 (ddd, J = 7.3, 4.4, 1.5 Hz, 3H) 7.48 (td, J =
7.8, 2.9 Hz, 3H) 7.53 (d, J = 1.0 Hz, 1H) 7.32 (d, J = 1.0 Hz, 1H)
7.25−7.21 (m, 1H) 4.23 (s, 2H) 4.19 (s, 3H) 3.98 (dd, J = 11.7, 2.9
Hz, 2H) 3.94−3.84 (m, 2H) 3.83−3.73 (m, 2H) 3.56 (td, J = 11.2,
2.4 Hz, 2H) 3.51−3.40 (m, 2H) 1.48 (s, 9H).
(R)-2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-N-(2-(morpholin-
2-yl)ethyl)isonicotinamide (51). TFA (0.032 mL, 0.411 mmol) was
added to a suspension of (R)-tert-butyl 2-(2-(2-benzyl-6-(1-methyl-
1H-1,2,3-triazol-4-yl)isonicotinamido)ethyl)morpholine-4-carboxylate
(75, 104 mg, 0.205 mmol) in DCM (3 mL), and the mixture was
stirred at rt under nitrogen for 3 h after which further TFA (0.032 mL,
0.411 mmol) was added. Stirring continued for 2 h after which further
TFA (0.032 mL, 0.411 mmol) was added, and the reaction mixture
was left to stand in solution overnight. The reaction mixture was
quenched with sat. sodium bicarbonate solution (10 mL) and
extracted with DCM (3 × 20 mL). The organic layer was passed
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through a hydrophobic frit, and the solvent was removed under
reduced pressure. The resulting oil was dissolved in DCM and
purified by silica chromatography using a gradient of 0−16% 2 M
methanolic ammonia/DCM. The product-containing fractions were
combined, and the solvent was removed in vacuo. The product was
left to dry in vacuo overnight to give (R)-2-benzyl-6-(1-methyl-1H-
1,2,3-triazol-4-yl)-N-(2-(morpholin-2-yl)ethyl)isonicotinamide (51,
28.6 mg, 0.070 mmol, 34% yield) as a pale yellow solid. LC−MS
solution was then stirred for 30 min at rt, benzyl alcohol (6.50 mL,
62.5 mmol) was added, and the mixture was heated at reflux for 3 h.
The reaction mixture was diluted with EtOAc (100 mL) and washed
with water (100 mL), the organic layer was dried and evaporated in
vacuo, and the residue was purified by silica chromatography eluting
with 0−50% EtOAc/cyclohexane. Product-containing fractions were
combined and evaporated in vacuo to give (R)-tert-butyl 3-
(2(((benzyloxy)carbonyl)amino)ethyl)-3-fluoropiperidine-1-carboxy-
late (134, 8.9 g, 23.39 mmol, 75% yield) as a colorless gum. LC−MS
1
(high pH, ES+) tR = 0.83 min; m/z = 407.4; (100% pure) H NMR
1
(400 MHz, DMSO-d6): δ 8.84 (t, J = 5.4 Hz, 1H) 8.58 (s, 1H) 8.25
(d, J = 1.5 Hz, 1H) 7.56 (d, J = 1.5 Hz, 1H) 7.36−7.29 (m, 4H) 7.22
(tt, J = 6.4, 2.0 Hz, 1H) 4.18 (s, 2H) 4.13 (s, 3H) 3.71 (dt, J = 9.8, 2.0
Hz, 1H) 3.46−3.34 (m, 4H, obscured by water) 2.77 (dd, J = 12.2,
2.4 Hz, 1H), 2.65 (br s, 2H) 2.35 (dd, J = 12.2, 10.3 Hz, 1H) 1.60 (q,
J = 7.3 Hz, 2H).
(formic, ES+) tR = 1.22 min; m/z = 381.3; H NMR (400 MHz,
DMSO-d6): δ 7.39−7.28 (m, J = 8.3, 5.9 Hz, 4H) 7.24 (t, J = 5.4 Hz,
1H) 5.02 (s, 2H) 3.74 (br d, J = 12.7 Hz, 2H) 3.18−3.11 (m, J = 6.8
Hz, 2H) 3.07−2.93 (m, 1H) 2.87 (br t, J = 10.0 Hz, 1H) 1.89−1.80
(m, 1H) 1.79−1.53 (m, 4H) 1.51−1.44 (m, 1H) 1.39 (s, 9H).
(R)-tert-Butyl 3-(2-Aminoethyl)-3-fluoropiperidine-1-carboxylate
(135). (R)-tert-Butyl 3-(2-(((benzyloxy)carbonyl)amino)ethyl)-3-flu-
oropiperidine-1-carboxylate (134, 8.9 g, 23.39 mmol) was dissolved in
ethanol (100 mL), added to Pd/C (2 g) under vacuum, and then
hydrogenated at atmospheric pressure for 48 h. The mixture was
filtered through celite under nitrogen, and the filtrate was evaporated
in vacuo to give (R)-tert-butyl 3-(2-aminoethyl)-3-fluoropiperidine-1-
carboxylate (135, 6.0 g, 24.36 mmol, 104% yield) as a colorless oil. 1H
NMR (400 MHz, DMSO-d6): δ 3.75 (dt, J = 13.2, 3.9 Hz, 1H) 3.86−
3.71 (m, 1H) 3.13−3.05 (m, 1H) 2.92−2.80 (m, 1H) 2.70−2.65 (m,
J = 7.3 Hz, 2H) 1.87−1.77 (m, 1H) 1.72−1.53 (m, 4H), 1.50−1.43
(m, 1H), 1.39 (s, 9H).
(S)-tert-Butyl 2-(2-(2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamido)ethyl)morpholine-4-carboxylate (76). General pro-
cedure C was followed using the following amounts: DIPEA (83 μL,
0.476 mmol), 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic
acid (82, 70 mg, 0.238 mmol), (S)-tert-butyl 2-(2-aminoethyl)-
morpholine-4-carboxylate (123, 54.8 mg, 0.238 mmol), HATU (90
mg, 0.238 mmol), and DMF. The sample was purified by silica
chromatography using a gradient of 33−100% EtOAc/cyclohexane.
The product-containing fractions were combined, and the solvent was
removed in vacuo to give (S)-tert-butyl 2-(2-(2-benzyl-6-(1-methyl-
1H-1,2,3-triazol-4-yl)isonicotinamido)ethyl)morpholine-4-carboxylate
(76, 108 mg, 0.185 mmol, 78% yield) as a pale yellow solid. LC−MS
(R)-tert-Butyl 3-(2-(2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamido)ethyl)-3-fluoropiperidine-1-carboxylate (77). Gen-
eral procedure C was followed using the following amounts: DIPEA
(0.089 mL, 0.510 mmol), 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-
yl)isonicotinic acid (82, 75 mg, 0.255 mmol), (R)-tert-butyl 3-(2-
aminoethyl)-3-fluoropiperidine-1-carboxylate (135, 82 mg, 0.331
mmol), HATU (126 mg, 0.331 mmol), and DMF (2 mL). The
sample was purified by silica chromatography using a gradient of 30−
100% EtOAc/cyclohexane. The product-containing fractions were
combined, and the solvent was removed in vacuo to give (R)-tert-butyl
3-(2-(2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinamido)-
ethyl)-3-fluoropiperidine-1-carboxylate (77, 102 mg, 0.176 mmol,
69% yield) as a pale yellow solid. LC−MS (formic, ES+) tR = 1.17
1
(formic, ES+) tR = 1.13 min; m/z = 507.4; H NMR (400 MHz,
CDCl3-d): δ 8.18 (s, 2H) 7.55−7.50 (m, 2H) 7.34−7.30 (m, 4H)
4.23 (s, 2H) 4.20 (s, 3H) 4.01−3.96 (m, 2H) 3.84−3.75 (m, 2H)
3.56 (td, J = 11.7, 2.4 Hz, 2H) 3.49−3.41 (m, 2H) 1.87−1.71 (m,
3H) 1.48 (s, 9H).
(S)-2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-N-(2-(morpholin-
2-yl)ethyl)isonicotinamide (52). TFA (0.246 mL, 3.20 mmol) was
added to a suspension of (S)-tert-butyl 2-(2-(2-benzyl-6-(1-methyl-
1H-1,2,3-triazol-4-yl)isonicotinamido)ethyl)morpholine-4-carboxylate
(76, 108 mg, 0.213 mmol) in DCM (2 mL), and the reaction mixture
was stirred at rt for 5 h before the solvent was removed in vacuo. The
resulting solid was dissolved in MeOH and purified using a 5 g Isolute
SCX-2 cartridge using methanol followed by 2 M methanolic
ammonia. The basic fractions were combined, and the solvent was
removed in vacuo. The product was left to dry in vacuo for 90 min to
give (S)-2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-N-(2-(morpho-
lin-2-yl)ethyl)isonicotinamide (52, 57 mg, 0.140 mmol, 66% yield)
as a white solid. LC−MS (formic, ES+) tR = 0.57 min; m/z = 407.4;
(100% pure) 1H NMR (400 MHz, DMSO-d6): δ 8.89 (t, J = 5.4 Hz,
1H) 8.60 (s, 1H) 8.26 (d, J = 1.5 Hz, 1H) 7.57 (d, J = 1.5 Hz, 1H)
7.36−7.28 (m, 4H) 7.22 (tt, J = 6.8, 2.0 Hz, 1H) 4.17 (s, 2H) 4.13 (s,
3H) 3.74 (dd, J = 11.7, 2.0 Hz, 1H) 3.48−3.24 (obs. m, 7H) 2.81 (dd,
J = 12.2, 1.5 Hz, 1H) 2.68−2.60 (m, J = 11.2, 11.2, 3.4 Hz, 1H) 2.38
(dd, J = 12.2, 10.3 Hz, 1H) 1.60 (br q, J = 7.3 Hz, 2H).
1
min; m/z = 523.2; H NMR (400 MHz, CDCl3-d): δ 8.21−8.16 (m,
2H) 7.51 (d, J = 1.5 Hz, 1H) 7.31 (obs. s, 5H) 4.23 (s, 2H) 4.20 (s,
3H) 3.74−3.60 (m, 4H) 2.03−1.88 (m, 4H) 1.87−1.76 (m, 2H)
1.74−1.63 (m, 2H) 1.46 (s, 9H).
(R)-2-Benzyl-N-(2-(3-fluoropiperidin-3-yl)ethyl)-6-(1-methyl-1H-
1,2,3-triazol-4-yl)isonicotinamide (53). TFA (0.150 mL, 1.952
mmol) was added to a suspension of (R)-tert-butyl 3-(2-(2-benzyl-
6-(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinamido)ethyl)-3-fluoropi-
peridine-1-carboxylate (77, 102 mg, 0.195 mmol) in DCM (3 mL).
The reaction mixture was stirred at rt under nitrogen for 1 h and left
to stand in solution overnight. The reaction mixture was concentrated
in vacuo and purified using a 5 g Isolute SCX-2 cartridge using
sequential methanol followed by 2 M methanolic ammonia. The basic
fractions were combined, and the solvent was removed in vacuo. The
impure product was dissolved in 1:1 DMSO/MeOH and purified by
MDAP (high pH). The product-containing fraction was concentrated
and dried in vacuo for 2 h to give (R)-2-benzyl-N-(2-(3-
fluoropiperidin-3-yl)ethyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (53, 36.4 mg, 0.086 mmol, 44% yield) as a white
solid. LC−MS (high pH, ES+) tR = 0.91 min; m/z = 423.4; (100%
(R)-3-(1-(tert-Butoxycarbonyl)-3-fluoropiperidin-3-yl)propanoic
Acid (133). (R)-tert-Butyl 3-(3-ethoxy-3-oxopropyl)-3-fluoropiperi-
dine-1-carboxylate (125, 9.6 g, 31.6 mmol) was dissolved in ethanol
(50 mL), and NaOH (47.5 mL, 95 mmol) was added. The solution
was then stirred at rt for 4 h. The solvent was evaporated in vacuo and
the residue was partitioned between water (100 mL) and ether (100
mL). The aqueous layer was acidified with 2 M HCl to pH 2 and then
extracted with EtOAc (2 × 100 mL). The organic layer was washed
with water (100 mL), then dried, and evaporated in vacuo to give (R)-
3-(1-(tert-butoxycarbonyl)-3-fluoropiperidin-3-yl)propanoic acid
1
pure) H NMR (400 MHz, DMSO-d6): δ 8.90 (t, J = 5.4 Hz, 1H)
8.60 (s, 1H) 8.25 (d, J = 1.5 Hz, 1H) 7.56 (d, J = 1.5 Hz, 1H) 7.36−
7.28 (m, 4H) 7.22 (tt, J = 6.8, 2.0 Hz, 1H) 4.17 (s, 2H) 4.13 (s, 3H)
2.82−2.66 (m, 3H), 2.61 (t, J = 13.2 Hz, 1H) 2.23−2.05 (m, 1H)
1.91−1.76 (m, 3H) 1.73−1.62 (m, 1H) 1.62−1.51 (m, 2H) 1.44−
1.36 (m, 1H).
1
(133, 8.6 g, 31.2 mmol, 99% yield) as a colorless solid. H NMR
(400 MHz, CDCl3-d): δ 3.76 (dt, J = 13.2, 4.4 Hz, 1H) 3.06 (br d, J =
9.3 Hz, 2H) 2.55 (t, J = 7.8 Hz, 2H) 2.04−1.88 (m, 3H) 1.86−1.73
(m, 1H) 1.68−1.50 (m, 3H) 1.47 (s, 9H).
(R)-tert-Butyl 3-(2-(((Benzyloxy)carbonyl)amino)ethyl)-3-fluoro-
piperidine-1-carboxylate (134). Diphenyl phosphorazidate (8.08
mL, 37.5 mmol) was added to a mixture of (R)-3-(1-(tert-
butoxycarbonyl)-3-fluoropiperidin-3-yl)propanoic acid (133, 8.6 g,
31.2 mmol) and Et3N (13.06 mL, 94 mmol) in toluene (50 mL). The
2-Benzyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-N-(3-(piperidin-4-yl)-
propyl)isonicotinamide (54). General procedure C was followed
using the following amounts: 2-benzyl-6-(1-methyl-1H-1,2,3-triazol-4-
yl)isonicotinate, sodium salt (132, 0.032 g, 0.1 mmol), HATU (0.038
g, 0.100 mmol), DMF (0.5 mL), DIPEA (0.055 mL, 0.315 mmol),
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and 3-(piperidin-4-yl)propan-1-amine (0.120 mmol). The reaction
mixture was capped, shaken, and left to stand at rt for 2 h. HATU (20
mg) was added to the reaction mixture with DIPEA (30 μL). The
vessel was capped, shaken, and left to stand at rt for 18 h. A solution
of T3P 50% in EtOAc (120 μL) was added to the reaction vial, and
DIPEA (55 μL) was also added. The reaction mixture was capped,
shaken, and stood at rt for 1 h. The solvent was removed. The sample
was dissolved in DMSO (2 mL) and purified by high pH MDAP. The
solvent was dried under a stream of nitrogen in the Radleys blowdown
apparatus. To the reaction mixture were added DCM (0.5 mL) and
TFA (0.5 mL). The mixture was then capped, shaken, and left to
stand at rt for 2 h. The solvent was removed to dryness. An SCX-2
SPE column [0.5 g, preconditioned with MeOH (1 mL)] was
prepared, and the sample was dissolved in MeOH (0.5 mL) and
added to the column, before it was washed thoroughly with a further
MeOH (1 mL). The column was finally washed thoroughly with 2 M
NH3 in MeOH (1 mL) and these final washings only were collected.
The solvent was removed to dryness to give 2-benzyl-6-(1-methyl-1H-
1,2,3-triazol-4-yl)-N-(3-(piperidin-4-yl)propyl)isonicotinamide (54,
7.3 mg, 0.017 mmol, 16% yield). LC−MS (formic, ES+) tR = 0.58
compound was characterized by mass spectroscopy and analytical
reversed-phase HPLC.
Compounds were titrated from 10 mM in 100% DMSO and 50 nL
was transferred to a low volume black 384-well microtitre plate using
a Labcyte Echo 555. A Thermo Scientific Multidrop Combi was used
to dispense 5 μL of 20 nM protein in an assay buffer of 50 mM
HEPES, 150 mM NaCl, 5% glycerol, 1 mM DTT, and 1 mM CHAPS,
pH 7.4, and in the presence of 100 nM fluorescent ligand (∼Kd
concentration for the interaction between BRD4 BD1 and ligand).
After equilibrating for 30 min in the dark at rt, the bromodomain
protein/fluorescent ligand interaction was detected using TR-FRET
following a 5 μL addition of 3 nM europium chelate-labeled anti-6His
antibody (PerkinElmer, W1024, AD0111) in assay buffer. Time-
resolved fluorescence (TRF) was then detected on a TRF laser-
equipped PerkinElmer Envision multimode plate reader (excitation =
337 nm; emission 1 = 615 nm; emission 2 = 665 nm; dual wavelength
bias dichroic = 400 nm, 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 high (DMSO) and low (compound control
derivative of I-BET762) controls and IC50 values were determined for
each of the compounds tested by fitting the fluorescence ratio data to
a four-parameter model
1
min; m/z = 419.3; (100% pure) H NMR (400 MHz, DMSO-d6): δ
8.84 (t, J = 5.5 Hz, 1H) 8.58 (s, 1H) 8.25 (d, J = 1.5 Hz, 1H) 7.57 (d,
J = 1.5 Hz, 1H) 7.37−7.28 (m, 4H) 7.22 (tt, J = 6.5, 2.0 Hz, 1H) 4.18
(s, 2H) 4.13 (s, 3H) 3.58−3.33 (m, 2H) 3.25 (td, J = 7.1, 6.0 Hz, 2H)
2.96 (dt, J = 12.1, 3.5 Hz, 2H) 1.62 (br d, J = 12.1 Hz, 2H) 1.57−1.50
(m, 2H) 1.39−1.28 (m, 1H) 1.26−1.18 (m, 2H) 1.02 (qd, J = 12.1,
4.5 Hz, 2H).
y = A + (B − A)/(1 + (10ĉ /x)̂D)
where “A” is the minimum, “B” is the Hill slope, “c” is the IC50, and
“D” is the maximum.
(S)-2-Benzyl-N-(3-(3-fluoropiperidin-3-yl)propyl)-6-(1-methyl-
1H-1,2,3-triazol-4-yl)isonicotinamide (55). A solution of 2-benzyl-6-
(1-methyl-1H-1,2,3-triazol-4-yl)isonicotinic acid (82, 0.029 g, 0.1
mmol) and HATU (0.038 g, 0.100 mmol) dissolved in DMF (0.5
mL) was prepared. DIPEA (0.055 mL, 0.315 mmol) was added before
the mixture was shaken and added to preweighed (R)-3-(3-
fluoropiperidin-3-yl)propan-1-amine (0.120 mmol). The reaction
mixture was capped, shaken, and left to stand at rt for 18 h. The
sample, in DMF, was taken up into DMSO (0.5 mL) and purified by
high pH MDAP. The solvent was dried under a stream of nitrogen in
the Radleys blowdown apparatus. To the concentrate were added
DCM (0.5 mL) and TFA (0.5 mL). The mixture was capped, shaken,
and left to stand at rt for 2 h, before the solvent was removed to
dryness. An SCX-2 SPE column [0.5 g, preconditioned with MeOH
(1 mL)] was prepared and the sample dissolved in MeOH (0.5 mL)
was added. The column was washed with MeOH (1 mL) followed by
2 M NH3 in MeOH (1 mL). The basic washes were collected, and the
solvent was removed to dryness to give (S)-2-benzyl-N-(3-(3-
fluoropiperidin-3-yl)propyl)-6-(1-methyl-1H-1,2,3-triazol-4-yl)-
isonicotinamide (55, 7.1 mg, 0.016 mmol, 15% yield). LC−MS
(formic, ES+) tR = 0.58 min; m/z = 437.2; (95% pure) 1H NMR (600
MHz, DMSO-d6): δ 8.91 (t, J = 5.3 Hz, 1H) 8.60−8.59 (m, 1H)
8.27−8.26 (m, 1H) 7.58−7.57 (m, 1H) 7.36−7.30 (m, J = 7.2 Hz,
4H) 7.22 (t, J = 7.2 Hz, 1H) 4.18 (s, 2H) 4.13 (s, 3H) 2.98−2.92 (m,
2H) 2.62−2.55 (m, 2H) 1.84−1.78 (m, 2H) 1.65−1.59 (m, J = 13.6
Hz, 6H) 1.53−1.47 (m, 2H) 1.44−1.38 (m, 1H).
Physicochemical Properties. Permeability across a lipid
membrane, chromatographic logD at pH 7.4, and CLND solubility
by precipitation into saline were measured using published
protocols.65,74−76
FaSSIF Solubility. This experiment determines the solubility of
solid compounds in FaSSIF at pH 6.5 after 4 h equilibration at room
temperature. A total of 1 mL of FaSSIF buffer (3 mM Sodium
taurocholate, 0.75 mM lecithin in sodium phosphate buffer at pH 6.5)
is added to manually weighed 1 mg of the solid compound in a 4 mL
vial.77 The resulting suspension is shaken at 900 rpm for 4 h at room
temperature and then transferred to a Multiscreen HTS, 96-well
solubility filter plate. The residual solid is removed by filtration. The
supernatant solution is quantified by HPLC-UV using single-point
calibration of a known concentration of the compound in DMSO.
The dynamic range of the assay is 1−1000 μg/mL.
All 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 Animals.
The human biological samples were sourced ethically and their
research use was in accordance with the terms of the informed
consents under an IRB/EC-approved protocol.
Intrinsic Clearance (CLint) Measurements. The human bio-
logical samples were sourced ethically and their research use was in
accordance with the terms of the informed consents under an IRB/
EC-approved protocol.
BRD4 Mutant TR-FRET Assay.73 Tandem bromodomains of
6His-Thr-BRD4(1−477) were expressed, with an appropriate
mutation in BD2 (Y390A) to monitor compound binding to BD1
or in BD1 (97A) to monitor compound binding to BD2. Analogous Y
→ A mutants were used to measure binding to the other BET
bromodomains: 6His-Thr-BRD2 (1−473 Y386A or Y113A), 6His-
Thr-BRD3 (1−435 Y348A or Y73A), and 6His-FLAG-Tev-BRDT
(1−397 Y309A or Y66A). The AlexaFluor 647-labeled BET
bromodomain ligand was prepared as follows: to a solution of
AlexaFluor 647 hydroxysuccinimide ester in DMF was added a 1.8-
fold excess of N-(5-aminopentyl)-2-((4S)-6-(4-chlorophenyl)-8-me-
thoxy-1-methyl-4H-benzo[f ][1,2,4]triazolo[4,3-a][1,4]-diazepin-4-
yl)acetamide, also in DMF, and when thoroughly mixed, the solution
was basified by the addition of a threefold excess of diisopropylethyl-
amine. Reaction progress was followed by electrospray LC/MS, and
when judged completely, the product was isolated and purified by
reversed-phase C18 high-performance LC (HPLC). The final
Microsome intrinsic clearance data were determined by Cyprotex
UK. To test the metabolic stability of 12, it was incubated in male
Wistar Han rat and mixed gender-pooled human liver microsomes.
Microsomes (final protein concentration 0.5 mg/mL), 0.1 M
phosphate buffer pH 7.4, and test compound (final substrate
concentration = 0.5 μM) were preincubated at 37 °C prior to the
addition of NADPH (final concentration = 1 mM) to initiate the
reaction. The test compound was incubated for 0, 5, 15, 30, and 45
min. The control (minus NADPH) was incubated for 45 min only.
The reactions were stopped by the addition of 50 μL of methanol
containing the internal standard at the appropriate time points.
Following protein precipitation, the compound remaining in the
supernatants was measured using specific LC−MS/MS methods as a
ratio to the internal standard in the absence of a calibration curve.
Peak area ratios (compound to IS) were fitted to an unweighted
logarithmic decline in the substrate. Using the first-order rate
constant, clearance was calculated by adjustment for protein
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concentration, volume of the incubation, and hepatic scaling factor
(52.5 mg microsomal protein/g liver for all species).
Rat Surgical Preparation for the IV Infusion Study. Male
Wistar Han rats (supplied by Charles River UK Ltd.) were surgically
prepared at GSK with implanted cannulae in the femoral vein (for
drug administration) and jugular vein (for blood sampling). The rats
received cefuroxime (116 mg/kg sc) and carprofen (7.5 mg/kg sc) as
a preoperative antibiotic and analgesic, respectively. The rats were
allowed to recover for at least 2 days prior to dosing and had free
access to food and water throughout.
Hepatocyte intrinsic clearance data were determined by Cyprotex
UK. The test compound (0.5 μM) was incubated with cryopreserved
hepatocytes in suspension. Samples were removed at 6 time points
over the course of a 60 min (rat) or 120 min (human) experiment,
and the test compound was analyzed by LC−MS/MS. Cryopreserved
pooled hepatocytes were purchased from a reputable commercial
supplier and stored in liquid nitrogen prior to use. Williams E media
was supplemented with 2 mM L-glutamine and 25 mM HEPES, and
the test compound (final substrate concentration 0.5 μM; final
DMSO concentration 0.25%) was preincubated at 37 °C prior to the
addition of a suspension of cryopreserved hepatocytes (final cell
density 0.5 × 106 viable cells/mL in Williams E media supplemented
with 2 mM L-glutamine and 25 mM HEPES) to initiate the reaction.
The final incubation volume was 500 μL. The reactions were stopped
by transferring 50 μL of the incubate to 100 μL of acetonitrile at the
appropriate time points. The termination plates were centrifuged at
2500 rpm at 4 °C for 30 min to precipitate the protein. The remaining
incubate (200 μL) was crashed with 400 μL of acetonitrile at the end
of the incubation. Following protein precipitation, the sample
supernatants were combined in cassettes of up to 4 compounds and
analyzed using Cyprotex generic LC−MS/MS conditions.
Rat IV n = 1 PK Study. Surgically prepared male Wistar Han rats
received a 1 h intravenous (iv) infusion of compound 28, 36, or 29 as
a discrete dose, formulated in DMSO and 10% (w/v) Kleptose HPB
in saline aq [2:98% (v/v)] at a concentration of 0.2 mg/mL to
achieve a target dose of 1 mg/kg. Serial blood samples (25 μL) were
collected predose and up to 7 h after the start of the iv infusion.
Diluted blood samples were analyzed for the parent compound using
a specific LC−MS/MS assay (LLQ = 1−2 ng/mL). At the end of the
study, the rats were euthanized using a Schedule 1 technique.
̈
Rat PO n = 3 PK Study. Three naive male Wistar Han rats with
no surgical preparation received an oral gavage administration of
compound 28, 36, or 29 as a discrete dose, suspended in 1% (w/v)
methylcellulose aq at a concentration of 0.6 mg/mL to achieve a
target dose of 3 mg/kg. Serial blood samples (25 μL) were collected
via temporary tail vein cannulation up to 24 h after oral dosing and
additional blood sampling via tail vein venepuncture up to 24 h after
oral dosing. Diluted blood samples were analyzed for the parent
compound using a specific LC−MS/MS assay (LLQ = 1 ng/mL). At
the end of the study, the rats were euthanized using a Schedule 1
technique.
Intrinsic Clearance (CLint) Data Analysis. From a plot of ln
peak area ratio (compound peak area/internal standard peak area)
against time, the gradient of the line was determined. Subsequently,
half-life (t1/2) and intrinsic clearance (CLint) were calculated using the
equations given below
Blood Sample Analysis. Diluted blood samples (1:1 with water)
were extracted using protein precipitation with acetonitrile containing
an analytical internal standard. An aliquot of the supernatant was
analyzed by reverse-phase LC−MS/MS using a heat-assisted
electrospray interface in the positive ion mode. Samples were assayed
against calibration standards prepared in control blood.
elimination rate constant (k) = (−gradient)
0.693
half‐life (t1/2) (min) =
k
PK Data Analysis from PK Studies. PK parameters were
obtained from the blood concentration−time profiles using non-
compartmental analysis with WinNonlin Professional 6.3 (Pharsight,
Mountain View, CA).
V × 0.693
intrinsic clearance (CLint)(μL/min /million cells) =
t1/2
where V = incubation volume (μL)/number of cells.
hWB MCP-1 Assay. The human biological samples were sourced
ethically and their research use was in accordance with the terms of
the informed consents under an IRB/EC-approved protocol.
Compounds to be tested were diluted in 100% DMSO to give a
range of appropriate concentrations at 140×, the required final assay
concentration, of which 1 μL was added to a 96-well tissue culture
plate. A total of 130 μL of hWB, collected into a sodium heparin
anticoagulant (1 unit/mL final), was added to each well and plates
were incubated at 37 °C (5% CO2) for 30 min before the addition of
10 μL of 2.8 μg/mL LPS (Salmonella typhosa), diluted in complete
RPMI 1640 (final concentration 200 ng/mL), to give a total volume
of 140 μL per well. After further incubation for 24 h at 37 °C, 140 μL
of phosphate-buffered saline was added to each well. The plates were
sealed, shaken for 10 min, and then centrifuged (2500 rpm × 10 min).
A total of 100 μL of the supernatant was removed and MCP-1 levels
were assayed immediately by immunoassay (MesoScale Discovery
technology).
Fraction Unbound in Blood. Control blood from Wistar Han
rats was obtained on the day of experimentation from in-house GSK
stock animals. The fraction unbound in blood was determined using a
rapid equilibrium dialysis (RED) technology plate (Linden Bio-
science, Woburn, MA) at a concentration of 200 and 1000 ng/mL.
Blood was dialyzed against phosphate-buffered saline solution by
incubating the dialysis units at 37 °C for 4 h. Following incubation,
aliquots of blood and buffer were matrix-matched prior to analysis by
LC−MS/MS. The unbound fraction was determined using the peak
area ratios in buffer and in blood as a mean value of the two
concentrations investigated.
Human Serum Albumin Measurements. Chemically bonded
human serum albumin (HSA) columns, with the dimensions of 50
mm × 3 mm id, were obtained from Chromtech, Ltd (U.K.). The
mobile-phase flow rate was 1.8 mL/min. The starting mobile phase
was 50 mM aqueous ammonium acetate, with the pH adjusted to 7.4.
Mobile phase B was 100% 2-propanol (HPLC grade). The gradient
retention time of the compound was recorded using the following
gradient profile: 0−3 min linear gradient from 0 to 30% 2-propanol; 3
to 6 min constant mobile-phase composition of 30% 2-propanol; 6 to
6.5 min linear gradient back to 0% 2-propanol (100% ammonium
acetate buffer); and 6.5 to 10 min 100% ammonium acetate buffer.
In Vivo DMPK Studies. All 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
Animals.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02156.
X-ray crystallographic data and methods, DiscoverX
BROMOscan Bromodomain Profiling of 36, and LC−
MS and NMR spectra for 28, 36, and 39 (PDF)
For all in vivo studies, the temperature and humidity were
nominally maintained at 21 2 °C and 55 10%, respectively. The
diet for rodents was 5LF2 Eurodent Diet 14% (PMI Labdiet,
Richmond, IN). There were no known contaminants in the diet or
water at concentrations that could interfere with the outcome of the
studies.
Molecular formula strings (CSV)
Accession Codes
Authors will release the unpublished PDB ID, atomic
coordinates, and experimental data upon article publication.
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pubs.acs.org/jmc
Article
Robert J. Watson − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
James M. Woolven − Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Emmanuel H. Demont − Epigenetics Discovery Performance
Unit, GlaxoSmithKline, Medicines Research Centre,
Hertfordshire SG1 2NY, U.K.; orcid.org/0000-0001-
7307-3129
AUTHOR INFORMATION
■
Corresponding Author
Helen E. Aylott − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.; orcid.org/0000-0002-6362-1790;
Email: helen.x.aylott@gsk.com
Authors
Stephen J. Atkinson − Epigenetics Discovery Performance
Unit, GlaxoSmithKline, Medicines Research Centre,
Hertfordshire SG1 2NY, U.K.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02156
Paul Bamborough − Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.; orcid.org/0000-0001-9479-2894
Anna Bassil − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Chun-wa Chung − Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.; orcid.org/0000-0002-2480-3110
Laurie Gordon − Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Paola Grandi − IVIVT Cellzome, Platform Technology and
Science, GlaxoSmithKline, Heidelberg 69117, Germany
James R. J. Gray − Quantitative Pharmacology,
Immunoinflammation Therapy Area Unit, GlaxoSmithKline,
Medicines Research Centre, Hertfordshire SG1 2NY, U.K.
Lee A. Harrison − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.; orcid.org/0000-0003-1804-0687
Thomas G. Hayhow − Epigenetics Discovery Performance
Unit, GlaxoSmithKline, Medicines Research Centre,
Hertfordshire SG1 2NY, U.K.
Cassie Messenger − Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Darren Mitchell − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Alexander Phillipou − Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Alex Preston − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.; orcid.org/0000-0003-0334-0679
Rab K. Prinjha − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
All authors were GlaxoSmithKline full-time employees when
this study was performed.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Darrian Hollywood, Fiona Shilliday,
and other members of Platform Technology Sciences group at
GSK for protein reagent generation, assay, and crystallization
support; Tony Cooper and Heather Barnett for support with
chemistry arrays; Eric Hortense, Richard Briers, Steve Jackson,
and Sean Hindley for analytical and purification support; Sean
Lynn, Richard Upton, and Stephen Richards for assistance with
NMR analysis; and Matthew Bowen for assistance with
experimentals.
ABBREVIATIONS
■
AMP, artificial membrane permeability; BD1, bromodomain 1
(N-terminal bromodomain); BD2, bromodomain 2 (C-
terminal bromodomain); BET, bromo and extraterminal
domain; BRD2,3,4,T, bromodomain containing protein
2,3,4,T; CLb, blood clearance; CLint, intrinsic clearance;
CLND, chemiluminescent nitrogen detection; DIAD, diiso-
propyl azodicarboxylate; dppb, 1,4-bis(diphenylphosphino)-
butane; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide; EDG, electron-donating group; EWG, electron-with-
drawing group; FACS, flow cytometry staining buffer; fub,
fraction unbound in blood; HATU, 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
fluorophosphate; hWB, human whole blood; k, elimination
rate constant; KAc, acetylated lysine; LLEat, Astex lipophilic
ligand efficiency; MCP-1, monocyte chemoattractant protein-
1; MDAP, mass-directed auto preparation; MLA, mouse
lymphoma assay; STAB, sodium triacetoxyborohydride; V,
incubation volume; Vss, volume of distribution at the steady
state; WPF, tryptophan-proline-phenylalanine; 2-MeTHF, 2-
methyltetrahydrofuran
Francesco Rianjongdee − Epigenetics Discovery Performance
Unit, GlaxoSmithKline, Medicines Research Centre,
Hertfordshire SG1 2NY, U.K.
Inmaculada Rioja − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
Jonathan T. Seal − Epigenetics Discovery Performance Unit,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.; orcid.org/0000-0003-0148-5487
Ian D. Wall − Platform Technology and Science,
GlaxoSmithKline, Medicines Research Centre, Hertfordshire
SG1 2NY, U.K.
REFERENCES
■
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recognition and large-scale structural analysis of the human
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