GNE-371, a Potent and Selective Chemical Probe for the Second Bromodomains of Human Transcription-Initiation-Factor TFIID Subunit 1 and Transcription-Initiation-Factor TFIID Subunit 1-like

Article abstract of DOI:10.1021/acs.jmedchem.8b01225

The biological functions of the dual bromodomains of human transcription-initiation-factor TFIID subunit 1 (TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure-activity-relationship studies that ultimately led to our in vitro tool compound, 27 (GNE-371). Compound 27 binds TAF1(2) with an IC₅₀ of 10 nM while maintaining excellent selectivity over other bromodomain-family members. Compound 27 is also active in a cellular-TAF1(2) target-engagement assay (IC₅₀ = 38 nM) and exhibits antiproliferative synergy with the BET inhibitor JQ1, suggesting engagement of endogenous TAF1 by 27 and further supporting the use of 27 in mechanistic and target-validation studies.

Full text of DOI:10.1021/acs.jmedchem.8b01225

Article
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/jmc
GNE-371, a Potent and Selective Chemical Probe for the Second
Bromodomains of Human Transcription-Initiation-Factor TFIID
Subunit 1 and Transcription-Initiation-Factor TFIID Subunit 1‑like
†
†
,†
‡
†
Shumei Wang, Vickie Tsui, Terry D. Crawford,* James E. Audia, Daniel J. Burdick,
†
‡
‡
†
Maureen H. Beresini, Alexandre Cote, Richard Cummings, Martin Duplessis, E. Megan Flynn,
̂ ́
‡
‡
‡
§
‡
Michael C. Hewitt, Hon-Ren Huang, Hariharan Jayaram, Ying Jiang, Shivangi Joshi,
†
‡
‡
‡
†
Jeremy Murray, Christopher G. Nasveschuk, Eneida Pardo, Florence Poy, F. Anthony Romero,
‡
‡
§
§
‡
§
Yong Tang, Alexander M. Taylor, Jian Wang, Zhaowu Xu, Laura E. Zawadzke, Xiaoyu Zhu,
Brian K. Albrecht, Steven R. Magnuson, Steve Bellon, and Andrea G. Cochran
‡
†
‡
†
†
Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States
‡
Constellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, Massachusetts 02142, United States
§
Wuxi Apptec Company, Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, People’s Republic of China
S Supporting Information
*
ABSTRACT: The biological functions of the dual bromodomains of human transcription-initiation-factor TFIID subunit 1
TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe
(
the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal
structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure−activity-relationship studies
that ultimately led to our in vitro tool compound, 27 (GNE-371). Compound 27 binds TAF1(2) with an IC of 10 nM while
5
0
maintaining excellent selectivity over other bromodomain-family members. Compound 27 is also active in a cellular-TAF1(2)
target-engagement assay (IC50 = 38 nM) and exhibits antiproliferative synergy with the BET inhibitor JQ1, suggesting
engagement of endogenous TAF1 by 27 and further supporting the use of 27 in mechanistic and target-validation studies.
INTRODUCTION
not been identified to date. Herein, we report our discovery of
a TAF1(2)-bromodomain inhibitor, which will be a valuable
tool compound for investigations into the pharmacological
relevance of non-BET bromodomain proteins.
■
Bromodomains are protein modules that serve as epigenetic
readers. These modules, consisting of ∼110 amino acids,
specifically recognize acetylated lysine residues on histone tails
and play important roles in chromatin-structural changes and
TAF1 contains both a histone acetyltransferase (HAT)
1
−3
domain and a tandem bromodomain module, comprising the
transcriptional regulation.
Bromodomain-containing pro-
2
1
individual bromodomains TAF1(1) and TAF1(2). Although
the function of TAF1 has not been fully elucidated, it has been
teins have been implicated in cancer, inflammation, and viral
replication; therefore, it is important to understand the
potential phenotypes produced by inhibition of these
22
implicated in pathways associated with cancer. Recently,
Sdelci et al. showed that TAF1(2) synergizes with
bromodomain-containing protein 4 (BRD4) in controlling
4
−8
bromodomains.
Although a number of inhibitors of the
bromodomain and extra terminal (BET) family and non-BET
bromodomains have been disclosed recently,
9
−20
a highly
selective inhibitor of the second bromodomain of the human
Received: August 3, 2018
transcription-initiation-factor TFIID subunit 1 (TAF1(2)) has
©
XXXX American Chemical Society
A
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cancer-cell proliferation, making TAF1(2) a potentially
(Asn1583), which is also important for recognition of histone
acetyl lysine (KAc, Figure 1A). The gatekeeper residue,
Tyr1589, forms a π-stacking interaction with the pyrrolopyr-
23
25
promising oncology target. In addition, TAF1(2) is capable
of recognizing not only acetyl lysine but also butyryl and
crotonyl lysines, although the biological significance of this
idone; the 4-phenyl extends into a hydrophobic region called
24
27,28
unusual property of TAF1(2) is currently unknown. A potent
and selective inhibitor of TAF1(2) would help clarify the
TAF1(2) bromodomain function, which would not be possible
through full-protein-knockdown experiments. This inhibitor
may also aid the development of novel therapeutic agents.
Herein we report such an inhibitor of TAF1(2) that has
favorable biochemical properties, is potent in a cellular target-
engagement assay, and is suitable for use as a chemical probe
for TAF1(2).
the ZA channel,
interacting with residues that form a
28
lipophilic region known as the WPF motif, which includes
van der Waals contacts with Pro1527 and Phe1528 and an
2
6
edge-to-face interaction with Trp1526. The sterically
hindered amide positions the carbonyl out of plane, resulting
in a direct hydrogen bond to the backbone NH of Asn1533 of
the ZA channel. The dimethyl amine occupies an open space
above the WPF shelf that provides an opportunity for further
hydrophobic interactions by extending deeper into the pocket
(
Figure 1B).
RESULTS AND DISCUSSION
We also previously observed that the benzamide of
■
Recently, we reported modulation of the selectivity of a
compound 1 bound to BRD4(1) had a flipped conformation,
pyrrolopyridone scaffold for various bromodomains by varying
directed away from the BRD4(1) WPF shelf and instead into
25,26
25
the hydrophobic substituent on the pyridone nitrogen.
the ZA channel toward the solvent (Figure 2). The phenyl
Replacing the methyl on the pyrrolopyridone, 1, with a 1-
butenyl group (compound 2) exploited the ability of TAF1(2)
ring maintains hydrophobic contacts to the WPF shelf;
however, the π−π edge-to-face interaction is not observed,
nor is a hydrogen-bond interaction to the carbonyl. Never-
theless, the dimethyl substituents can form hydrophobic
contacts with the gatekeeper Ile146 to retain potency.
24,25
to bind to butyrylated and crotonylated lysine residues
and
greatly improved the selectivity over a small set of
bromodomains while maintaining TAF1(2) activity (Table
1
). This modest change to the inhibitor rearranged and
Initial attempts to improve potency for TAF1(2) targeted
substituents of the benzamide and the phenyl ring. From the
structure of compound 2 bound to TAF1(2), we hypothesized
that bulkier substitutions on the benzamide could lead to
increased potency by improving the hydrophobic protein−
surface interactions in that region (Figure 1B). We therefore
explored a limited set of benzamide substitutions on the phenyl
ring (Table 2). Replacement of the dimethyl amine with larger
hydrophobic substituents, such as diethyl amine (3),
pyrrolidine (4), or N-methylcyclohexylamine (5), had little
effect on TAF1(2) potency; however, these substituents did
result in a 3−5-fold erosion of selectivity against BRD4(1).
Introducing a basic amine (compounds 6 and 7) was
unfavorable for TAF1(2) potency and selectivity over
BRD4(1), BRD9, and especially CECR2. The morpholine
amide (8) at this position slightly increased TAF1(2) potency,
although BRD4(1) selectivity remained unaffected. From the
cocrystal structure of 8 bound to TAF1(2), we observe that the
morpholine ether does not make a direct interaction and
instead hydrogen-bonds to a water molecule approaching the
solvent interface. However, the ring optimally fills the
accessible volume in the lipophilic-shelf region, which may
account for the slight potency improvement (Figure 3A). As
the benzamide of compound 2 is flipped when bound to
BRD4(1), further amide substituents alone would be well-
tolerated by BRD4(1) and would not result in improved
selectivity for TAF1(2).
Table 1. Comparison of N-Butenyl (2) and N-Methyl (1)
Substitutions on the Pyridone Nitrogen of the
Pyrrolopyridone Core
stabilized a conserved water network in the binding site,
providing an excellent starting point for further optimization to
increase both potency against TAF1(2) while also improving
the bromodomain-selectivity profile, particularly against BET-
Having found an increase in potency with the morpholine
amide, we next explored disubstituted phenyl analogues. We
hypothesized that increased steric bulk might be well-tolerated
in the TAF1(2) ZA channel (Figure 3B), while also resulting
in unfavorable interactions within the smaller BRD4(1)
binding pocket (Figure 2). The 5-fluoro (9) and 5-amino
(10) analogues showed no improvement in potency toward
TAF1(2), whereas 10 slightly lost selectivity against BRD4(1)
2
5
family members.
The N-butenyl group maintains potency against TAF1(2)
and improved selectivity against the other tested bromodo-
mains. All bromodomain assays were run in the TR-FRET
format as described in the Supporting Information. All IC50
data are reported as means of values from at least two
determinations.
(Table 3). Adding a larger OCHF substituent (11) showed a
2
The X-ray cocrystal structure of 2 in complex with the
second bromodomain of TAF1 (PDB code 5I1Q) reveals that
the pyrrolopyridone core makes a 2-point hydrogen-bond
interaction with the highly conserved asparagine residue
4-fold increase in potency compared with that of 8 but again
resulted in increased affinity for BRD4(1), whereas compound
12 (5-CN) was equipotent to 8 for TAF1(2) and BRD4(1).
However, both 11 and 12 were significantly more potent
B
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25
Figure 1. (A) Co-crystal structure of compound 2 (cyan) bound to TAF1(2) (PDB code 5I1Q, 1.5 Å resolution). TAF1(2) residues are in
yellow. The pyrrolopyridone core makes two strong hydrogen bonds with the conserved Asn1583, and the butenyl group extends into the water
channel. (B) Carbonyl of the benzamide forming a hydrogen bond with the NH of Asn1533.
25
Figure 2. (A) Compound 1 (magenta) bound to BRD4(1) (residues in salmon; PDB code 5I80, 1.5 Å resolution). (B) Protein surface in gray,
with the accessible region near the WPF shelf.
against CECR2, having submicromolar affinity. Substitution at
the 4-position with a fluorine (13), chlorine (14), methyl (15),
or 4,5-difluoro (16) provided at least a 5-fold decrease in
potency for BRD4(1), whereas the TAF1(2) potency was
maintained. We believe the improved selectivity for TAF1(2)
over BRD4(1) for compound 15 (142-fold) compared with
that of 8 (43-fold) may be the result of the methyl forcing the
benzamide orthogonal to the phenyl ring rather than at the 50°
angle observed for compound 1 in Figure 2A, resulting in a
negative interaction with the protein surface. Incorporation of
a pyridine nitrogen at position 5 on the phenyl ring
explored trisubstituted-phenyl-ring systems to induce steric
clashes in the BRD4 binding site. First we designed a number
of benzimidazole analogues to not only investigate steric effects
but also explore the potential of improving the edge-to-face
interaction with Trp1526. Compound 21 had similar potency
for TAF1(2) compared with that of 8, and had slightly
improved selectivity over BRD4(1) (Table 4). The N-methyl
regioisomers (22 and 23) had similar TAF1(2) potencies to
that of 21 and resulted in better selectivity over BRD4, but
their selectivity over CECR2 fell ∼6-fold. Increasing the steric
bulk with the ethyl compounds (24 and 25) did not result in
appreciable selectivity gains. We hypothesized that making the
benzimidazole regioisomer, 26, might introduce a hydrogen
bond with the gatekeeper Tyr1589 in TAF1(2). This could
both improve potency for TAF1(2) and also selectivity as a
result of modeled clashes with the sterically hindered β-
branched gatekeeper residues Ile146 and Val147 in BRD4(1)
and BRD4(2), respectively. Surprisingly, analogue 26 was
equipotent to compound 8 in the TAF1(2) assay, but perhaps
more importantly, 26 had significantly lower affinity for BRD4
and BRD9. We probed whether locking this compound in the
preferred tautomer would enable a stronger interaction to the
gatekeeper Try1589 and increase TAF1(2) potency. Com-
pound 27 resulted in 3-fold-improved TAF1(2) potency
compared with that of 26, further increased BRD4 selectivity
(>2000-fold), and improved selectivity for the other
bromodomains screened. The cocrystal structure of 27
confirms that the benzimidazole ring is indeed making a
hydrogen-bond interaction with Tyr1589 (Figure 4).
(
compound 17) improved TAF1(2) potency and also
provided improved selectivity over BRD4(1) (200-fold)
relative to that of 8 (43-fold). This is most likely due to the
flipped conformation of the benzamide in BRD4(1), where the
pyridine nitrogen is facing the lipophilic WPF-shelf region,
resulting in a desolvation penalty. Addition of a methyl (18) at
position 4 of the pyridine maintained potency against TAF1(2)
while increasing the selectivity window over BRD4(1)
(∼1250-fold). Investigation at the 4-position further with
methoxy (19) and ethoxy groups (20) greatly increased
TAF1(2) affinity, with single-digit nanomolar potency
observed. Unfortunately, these substitutions failed to further
improve selectivity over the bromodomain-family members in
our internal bromodomain panel.
Although compounds 19 and 20 met our initial goal of 100-
fold selectivity, further efforts continued with the aim of
obtaining an even greater selectivity window over BRD4 (IC50
>
20 μM) to ensure that any observed cellular phenotypes
were not compromised by BET inhibition. To achieve this, we
C
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Table 2. Structure−Activity Relationships of Phenylamides
a
All IC50 data are reported as means of values from at least two determinations.
Figure 3. (A) Cocrystal structure of compound 8 (orange) bound to TAF1(2) (PDB code 6DF4, 1.3 Å resolution). The morpholine amide
occupies significant volume on the WPF shelf. (B) Accessible volume extending from compound 8 off of the aryl ring.
The bromodomain selectivity of compound 27 was further
assessed by screening against 40 bromodomain-family
members in a BROMOscan panel (see Supporting Information
could be diminished by competition with the test compounds.
In this assay, 27 showed an IC50 of 0.038 μM (Figure 5A).
Compound selectivity was determined by measuring displace-
ment of a tracer compound from BRD4. BET inhibitor JQ1
demonstrated a potency of 0.3 μM, whereas 27 failed to
displace tracer even at the highest test concentration (20 μM,
Figure 5B), indicating a cellular selectivity of >500-fold for
TAF1(2) over BRD4, consistent with the high biochemical
selectivity observed.
29
Table S3 for full data profile). Exquisite selectivity for 27 was
observed, with only BRD4 (full length protein, KD = 8900
nM), CECR2 (K = 1200 nM), and BRD9 (K = 3400 nM)
D
D
potencies below 10 μM, whereas the potencies for TAF1(2)
and its homologue transcription-initiation-factor TFIID
subunit 1-like TAF1L(2) were very strong (1 and 5 nM,
respectively). Compound 27 was also screened at 1 μM in a
diverse panel of 35 kinases, with no inhibition >17% observed
Screening of a diverse panel of 393 cancer-cell lines for
sensitivity to 27 at different doses (up to 20 μM, 8 day assay)
revealed little to no effect on cell viability for any tested line
(not shown). However, positive interactions between TAF1
and BRD4 in mediating transcriptional activation have been
(
Table S4).
To confirm cellular permeability and assess the target-
engagement potential of 27, we made use of nanoBRET
3
0
23
assays. Treatment of cells transfected with nLuc−TAF1(2)
reported recently. In particular, a cellular screen for small
with a fluorescent, tagged ligand resulted in a BRET signal that
molecules capable of activating expression of a reporter gene
D
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Table 3. Structure-Activity Relationships of Morpholine Amides
a
All IC50 data are reported as means of values from at least two determinations.
sensitive to BRD4 inactivation yielded not only known BET-
bromodomain inhibitors as hits but also novel compounds,
some of which were not BET-bromodomain binders but
instead targeted TAF1(2). Synergistic loss of viability of H23
lung-cancer cells was observed when cells were treated with
combinations of JQ1 and a TAF1(2) inhibitor, CeMME13,
optimized for selectivity. Because of the higher potency of 27
compared with that of CeMME13, we carried out a similar
combination study with JQ1 and 27 to verify the earlier finding
deprotection or N-alkylation of the benzimidazole followed by
tosyl deprotection resulted in compounds 21−25.
The preparation of compounds 26 and 27 began with
reduction of methyl 4-amino-3-bromo-5-nitrobenzoate (38) to
the dianiline (39) with tin chloride (Scheme 4). Cyclization
with triethoxymethane afforded benzimidazole (40), which
was then converted to the boronic acid (41). Suzuki coupling
with 4-bromo-6-(but-3-en-1-yl)-1-tosyl-1H-pyrrolo[2,3-c]-
pyridin-7(6H)-one (28) yielded ester 42. Intermediate 42
was then either saponified to the acid followed by amide
coupling to yield compound 26 or alkylated with methyl iodide
to yield the desired N-methylbenzimidazole regioisomer (43)
as the major product. Saponification of 43 followed by amide
coupling led to compound 27.
23
(Figure 6). Enhanced sensitivity to JQ1 can be seen for
concentrations of 27 as low as ∼100 nM, consistent with the
potency of 27 in the target engagement assay. The drug
combination shows modest synergy in a Bliss model (overall
score = 15, maximum score = 43; Figure S1). The excellent
biochemical potency, selectivity, physical properties, and
observed cellular activity led us to designate compound 27
as GNE-371, a chemical probe for further biological evaluation
of TAF1 bromodomain 2.
CONCLUSION
■
In summary, we have identified a highly potent and selective in
vitro probe (27) of the TAF1(2) bromodomain through a
structure-based-design approach. On the basis of the potency
and exquisite selectivity profile and observed activity in cellular
engagement and phenotypic assays, compound 27 will be
suitable in interrogating the biology of TAF1(2) without
phenotypic contamination from BET inhibition.
Chemistry. Compounds 3−20 were made from the
25
previously reported pyrrolopyridone intermediate 28, either
via coupling of an aryl benzamide boronic acid or ester
(Scheme 1) or by Suzuki coupling to form the benzoic acid
intermediate followed by amide coupling (Scheme 2).
Preparation of compounds 21−25 began with an amide
coupling with 2-amino-3-nitrobenzoic acid (30) and morpho-
line to form 31 (Scheme 3). After bromination to afford 32
and iron reduction of the nitro group, the diamine (33) was
cyclized with triethoxymethane to give the benzimidazole (34).
After SEM protection and borylation, Suzuki coupling with 28
was followed by SEM deprotection with TFA. Either tosyl
EXPERIMENTAL PROCEDURES
■
General Methods. All solvents and reagents were used as
obtained. NMR analysis was performed in a deuterated solvent with a
Varian Avance 300 MHz or Bruker Avance 400 or 500 MHz NMR
spectrometer and referenced to trimethylsilane (TMS). Chemical
shifts are expressed as δ units using TMS as the external standard. The
E
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Table 4. Structure-Activity Relationships of Benzimidazoles
Figure 5. Cellular target-engagement assays. Each concentration point
was measured in triplicate and is shown as the mean ± SD. IC50
values were determined by fitting to a 4-parameter binding equation
in Prism 7 (GraphPad Software). (A) Compound 27 tested in a
nanoBRET TAF1(2) assay. The uncertainly in the IC50 is indicated
by the 95% confidence interval (CI) of the fit. (B) JQ1 and 27 tested
in a nanoBRET BRD4 assay (Promega N2131) using the full-length
nLuc−BRD4 construct supplied by the manufacturer. JQ1 IC in this
5
0
experiment is 0.36 μM.
on a CombiFlash Companion (Teledyne ISCO) with RediSep
normal-phase silica-gel (35−60 μm) columns and UV detection at
2
54 nm. Reverse-phase (HPLC) was used to purify compounds as
needed by elution from a Phenomenex Gemini-NX C18 column (20.2
50 mm, 5 μm) stationary phase, using the mobile phase indicated
×
and operating at a 35 mL/min flow rate on a Waters 3100 mass-
directed prep instrument. Chemical purities were >95% for all final
compounds as assessed by LC-MS analysis. The following analytical
method was used to determine chemical purities of final compounds:
an HPLC-Agilent 1200, water with 0.05% TFA, acetonitrile with
0.05% TFA (buffer B), and an Agilent SB-C18 (1.8 μM, 2.1 × 30
mm) were used at 25 °C with a gradient of 3−95% buffer B in 8.5 min
and 95% in 2.5 min (400 μL/min; 220 and 254 nm; Agilent
quadrupole 6140, ESI positive, 90−1300 amu).
a
All IC50 data are reported as means of values from at least two
Time-Resolved-Fluorescence-Resonance-Energy-Transfer
Assays. Compound potencies were evaluated in a panel of
biochemical bromodomain-binding assays. Binding of biotinylated
small-molecule ligands to recombinant His-tagged bromodomains was
assessed by time-resolved fluorescence-resonance-energy transfer
determinations.
following abbreviations were used in the NMR descriptions: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak. All
coupling constants (J) are reported in hertz. Mass spectra were
measured with a Finnigan SSQ710C spectrometer using an ESI
source coupled to a Waters 600MS high-performance-liquid-
chromatography (HPLC) system operating in reverse-phase mode
with an X-bridge Phenyl column of dimensions 150 × 2.6 mm, with 5
μm sized particles. Preparatory-scale silica-gel chromatography was
performed using medium-pressure liquid chromatography (MPLC)
24,25
(TR-FRET) as previously described.
3-(6-(But-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]-
pyridin-4-yl)-N,N-diethylbenzamide (3). To a solution of 4-
bromo-6-(but-3-en-1-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one
29 (45 mg, 0.17 mmol) and 3-(6-but-3-enyl-7-oxo-1H-pyrrolo[2,3-
c]pyridin-4-yl)-N,N-diethyl-benzamide (45 mg, 0.25 mmol) in
acetonitrile (1 mL), and 1 M potassium carbonate in water (1 mL)
was added Pd(dppf)Cl (14 mg, 0.02 mmol). The reaction was stirred
2
Figure 4. (A) Profile of compound 27 (GNE-371). All bromodomain assays were run in TR-FRET format. IC50 data are an average of at least two
independent experiments. LE = −RT ln(IC )/Nheavy atoms. LLE = pIC50 − cLogP. (B) Cocrystal structure of compound 27 (green) bound to
5
0
TAF1(2) (PDB code 6DF7, 2.0 Å resolution).
F
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Figure 6. H23 lung-cancer cells treated with combinations of JQ1 and 27. Viability was measured by luminescence after 48 h with CellTiter-Glo 2.0
reagent (Promega). Dose−response curves for indicated concentrations of 27 are shown on the left. An expanded set of the data from the same
experiment is shown in heatmap format on the right.
a
Scheme 1. Synthesis of Compounds 3−11, 13, 14, 17, and 20
a
Reagents and conditions: (i) MeOH, 10 M KOH solution, 50 °C, 77%; (ii) R-B(OH) , Pd(dppf)Cl , CH CN, 1 M K CO in water solution, 100
2
2
3
2
3
°C.
a
Scheme 2. Synthesis of Compounds 12, 15, 16, 18, and 19
a
Reagents and conditions: (i) R R -B(OH) , Pd(dppf)Cl , CH CN, 1 M K CO in water solution, 140 °C; (ii) R R NH, HATU, DIPEA, DMF.
1
2
2
2
3
2
3
3 4
at 100 °C for 10 min under microwave irradiation. The mixture was
filtered through a pad of Celite, using ethyl acetate (20 mL) to rinse
the cake. The filtrate was washed with brine (10 mL), dried over
sodium sulfate, and concentrated in vacuo. The residue was purified
by preparative HPLC (25−35% ACN/0.1% NH OH in H O) to
3-(6-(But-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]-
pyridin-4-yl)-N-cyclohexyl-N-methylbenzamide (5). Compound
was prepared in the same manner as shown for compound 3 except
3-(cyclohexyl(methyl)carbamoyl)phenyl)boronic acid was used
5
(
1
instead. H NMR (400 MHz, DMSO-d ) δ 12.13 (s, 1H), 7.70−
6
4
2
7
7
.62 (m, 1H), 7.56−7.48 (m, 2H), 7.43−7.34 (m, 2H), 7.30 (d, J =
.6 Hz, 1H), 6.41 (m, J = 2.9, 1.5 Hz, 1H), 5.86 (m, 1H), 5.14−4.98
afford 3-(6-(but-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]-
pyridin-4-yl)-N,N diethylbenzamide (17 mg, 28% yield) as a white
1
(m, 2H), 4.12 (m, 2H), 3.25 (s, 3H). 2.98−2.71 (m, 4H), 2.50−2.47
solid. H NMR (400 MHz, DMSO-d ) δ 12.13 (s, 1H), 7.72−7.61
6
(
4
m, 2H), 1.62 (d, J = 37.3 Hz, 4H), 1.02−0.90 (m, 2H). LC-MS: m/z
(
(
(
m, 1H), 7.57−7.48 (m, 2H), 7.41 (s, 1H), 7.38−7.27 (m, 2H), 6.42
+
04 (M+H) .
d, J = 2.8 Hz, 1H), 5.86 (ddt, J = 17.1, 10.2, 6.8 Hz, 1H), 5.13−4.97
6
-(But-3-en-1-yl)-4-(3-(4-methylpiperazine-1-carbonyl)-
m, 2H), 4.12 (dd, J = 7.9, 6.5 Hz, 2H), 3.45−3.20 (m, 4H), 2.48−
phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (6). Com-
pound 6 was prepared in the same manner as shown for compound 3
+
2
.40 (m, 2H), 1.12 (s, 6H). LC-MS: m/z 364 (M+H) .
6
-(But-3-en-1-yl)-4-(3-(pyrrolidine-1-carbonyl)phenyl)-1,6-
except (3-(4-methylpiperazine-1-carbonyl)phenyl)boronic acid was
dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (4). Compound 4 was
1
used instead. H NMR (400 MHz, DMSO-d ) δ 12.13 (s, 1H), 7.68
6
prepared in the same manner as shown for compound 3 except (3-
(
dt, J = 7.9, 1.3 Hz, 1H), 7.57−7.51 (m, 2H), 7.41 (s, 1H), 7.37 (d, J
2.8 Hz, 1H), 7.33 (dt, J = 7.6, 1.3 Hz, 1H), 6.42 (d, J = 2.8 Hz, 1H),
5.86 (m, 1H), 5.16−4.98 (m, 2H), 4.20−4.12 (m, 2H), 3.75−3.52
m, 4H), 2.41−2.26 (m, 6H), 2.20 (s, 3H). LC-MS: m/z 391 (M
1
(
pyrrolidine-1-carbonyl)phenyl)boronic acid was used instead.
H
=
NMR (400 MHz, DMSO-d ) δ 12.12 (s, 1H), 7.78 (dp, J = 3.8, 1.9
6
Hz, 1H), 7.72−7.65 (m, 2H), 7.58−7.33 (m, 3H), 6.43 (dd, J = 2.8,
(
+
+
1
2
3
.9 Hz, 1H), 5.86 (m, 1H), 5.13−4.98 (m, 2H), 4.12 (t, J = 7.3 Hz,
H), 3.46 (m, 4H), 2.50−2.46 (m, 2H), 1.86 (m, 4H). LC-MS: m/z
H) .
6-(But-3-en-1-yl)-4-(3-(4-(2-hydroxyethyl)piperazine-1-
carbonyl)phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one
+
62 (M+H) .
G
DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of Compounds 21−25
a
Reagents and conditions: (i) morpholine, HATU, DIPEA, DMF, 73%; (ii) bromine, acetic acid, 0 °C, 76%; (iii) Fe powder, NH Cl, EtOH, H O,
4
2
reflux, 92%; (iv) triethoxymethane, TsOH, rt, 76%; (v) SEM-Cl, NaH, DMF, 0 °C, 70%; (vi) bis(pinacolato)diboron, Pd(dppf)Cl , KOAc,
2
dioxane, 110 °C, 41% (crude); (vii) 28, Pd(dppf)Cl , Cs CO , dioxane, H O, 110 °C, 77%; (viii) NaOH, dioxane, H O, 80 °C, 5%; (ix) TFA,
2
2
3
2
2
DCM, rt, 98%; (x) NaH, R-I, DMF, 0 °C; (xi) NaOH, dioxane, H O, 80 °C.
2
(
7). Compound 7 was prepared in the same manner as shown for
except (3-fluoro-5-(morpholine-4-carbonyl)phenyl)boronic acid was
1
compound 3 except (3-(4-(2-hydroxyethyl)piperazine-1-carbonyl)-
phenyl)boronic acid was used instead. H NMR (400 MHz, DMSO-
d ) δ 12.14 (s, 1H), 7.68 (dt, J = 7.8, 1.4 Hz, 1H), 7.61−7.49 (m,
2
(
7
2
used instead. H NMR (400 MHz, DMSO-d ) δ 12.18 (s, 1H), 7.52
6
1
(s, 1H), 7.45 (t, J = 1.5 Hz, 1H), 7.40−7.36 (m, 2H), 7.23 (m, J = 8.8,
2.5, 1.3 Hz, 1H), 6.45 (d, J = 2.8 Hz, 1H), 6.00−5.75 (m, 1H), 5.21−
4.92 (m, 2H), 4.12 (t, J = 7.3 Hz, 2H), 3.62−3.20 (m, 8H), 2.49−
6
H), 7.46−7.30 (m, 3H), 6.42 (dd, J = 2.9, 1.6 Hz, 1H), 5.95−5.77
+
m, 1H), 5.14−4.97 (m, 2H), 4.48−4.42 (m, 1H), 4.30−4.12 (t, J =
2.45 (m, 2H). LC-MS: m/z 396 (M+H) .
.2 Hz, 2H), 3.70−3.62 (m, 2H), 3.45−3.39 (m, 2H), 2.45−2.38 (m,
4-(3-Amino-5-(morpholine-4-carbonyl)phenyl)-6-(but-3-en-
1-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (10). To a
solution of 4-bromo-6-but-3-enyl-1H-pyrrolo[2,3-c]pyridin-7-one
(29, 200 mg, 0.70 mmol) and [3-(morpholine-4-carbonyl)-5-nitro-
phenyl]boronic acid (300 mg, 0.90 mmol) in acetonitrile (2.5 mL)
and 1 M potassium carbonate in water (2.5 mL) was added
+
H). LC-MS: m/z 421 (M+H) .
6-(But-3-en-1-yl)-4-(3-(morpholine-4-carbonyl)phenyl)-1,6-
dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (8). Compound 8 was
prepared in the same manner as shown for compound 3 except (3-
(
1
morpholine-4-carbonyl)phenyl)boronic acid was used instead. H
NMR (400 MHz, DMSO-d ) δ 12.13 (s, 1H), 7.69 (dt, J = 7.9, 1.4
Pd(dppf)Cl (30 mg, 0.04 mmol). The mixture was stirred at 100 °C
2
6
Hz, 1H), 7.61−7.50 (m, 2H), 7.41 (s, 1H), 7.40−7.31 (m, 2H), 6.42
for 15 min under microwave irradiation. The mixture was filtered
through a pad of Celite and rinsed with ethyl acetate (20 mL). The
filtrate was washed with brine (10 mL), dried over sodium sulfate, and
concentrated in vacuo to yield 6-but-3-enyl-4-[3-(morpholine-4-
carbonyl)-5-nitro-phenyl]-1H-pyrrolo[2,3-c]pyridin-7-one (300 mg,
quant) as an off-white solid. This crude material was used in the
(
dd, J = 2.8, 1.9 Hz, 1H), 5.97−5.78 (m, 1H), 5.14−4.97 (m, 2H),
4
.12 (t, J = 7.2 Hz, 2H), 3.62−3.20 (m, 8H), 2.50−2.46 (m, 2H). LC-
+
MS: m/z 378 (M+H) .
6
-(But-3-en-1-yl)-4-(3-fluoro-5-(morpholine-4-carbonyl)-
phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (9). Com-
pound 9 was prepared in the same manner as shown for compound 3
+
next step without further purification. LC-MS: m/z 424 (M+H) .
H
DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Synthesis of Compound 27
a
Reagents and conditions: (i) SnCl ·2H O, ethyl acetate, 90 °C, 95%; (ii) triethoxymethane, TsOH·H O, THF, rt, 93%; (iii)
2
2
2
bis(pinanolato)diboron, Pd(dppf)Cl , KOAc, 1,4-dioxane, 120 °C, 92% (crude); (iv) Pd(dppf)Cl , Cs CO , 1,4-dioxane, water, 100 °C, 77%;
2
2
2
3
(
v) NaOH, methanol, water, 74%; (vi) morpholine, HATU, triethylamine, DMF, 21%; (vii) iodomethane, K CO , DMF, rt, 75%; (viii) NaOH,
2 3
methanol, water, 92%; (ix) morpholine, HATU, triethylamine, DMF, 33%.
To a mixture of 6-but-3-enyl-4-[3-(morpholine-4-carbonyl)-5-
nitro-phenyl]-1H-pyrrolo[2,3-c]pyridin-7-one (170 mg, 0.40 mmol)
in ethanol (8 mL) and water (3 mL) were added iron (220 mg, 4.0
mmol) and ammonium chloride (43 mg, 0.80 mmol). The reaction
was heated at 80 °C for 2 h. The mixture was filtered through a Celite
pad, the cake washed with ethanol, and the filtrate was concentrated
in vacuo. The residue was purified by preparative HPLC (25−35%
ACN/0.1% NH OH in H O) to give 3-(6-(but-3-en-1-yl)-7-oxo-6,7-
To a solution of (3-bromo-5-hydroxyphenyl)(morpholino)-
methanone (900 mg, 3.2 mmol) in DMF (20 mL)/H O (5 mL)
were added K CO (877 mg, 6.3 mmol) and sodium 2-chloro-2,2-
difluoroacetate (966 mg, 6.3 mmol). The reaction was heated at 100
C for 2 h. After cooling to ambient temperature, the mixture was
2
2
3
°
poured into water (600 mL), and the resulting mixture was extracted
with ethyl acetate (3 × 50 mL). The combined organic layers were
concentrated in vacuo. The residue was purified by flash
chromatography (1:1 petroleum ether/ethyl acetate) to give (3-
bromo-5-(difluoromethoxy)phenyl)(morpholino)methanone (400
4
2
dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-5-(morpholine-4-carbonyl)-
1
benzonitrile (37 mg, 23% yield) as a white solid. H NMR (400 MHz,
+
mg, 38% yield) as a yellow solid. LC-MS: m/z 338.1 (M+H) .
DMSO-d ) δ 12.06 (s, 1H), 7.35 (d, J = 2.8 Hz, 1H), 7.28 (s, 1H),
6
A mixture of (3-bromo-5-(difluoromethoxy)phenyl)(morpholino)-
methanone (400 mg, 1.19 mmol), bis(pinanolato)diboron(455 mg,
6
1
5
6
.89 (t, J = 1.9 Hz, 1H), 6.67 (t, J = 1.5 Hz, 1H), 6.53 (t, J = 1.8 Hz,
H), 6.42 (d, J = 2.8 Hz, 1H), 5.93−5.78 (m, 1H), 5.36 (s, 2H),
.12−4.96 (m, 2H), 4.10 (t, J = 7.2 Hz, 2H), 3.59 (s, 8H), 2.47 (d, J =
1
.79 mmol), potassium acetate (175 mg, 1.79 mmol), and
Pd(dppf)Cl (73 mg, 0.1 mmol) in dioxane (25 mL) was heated at
+
2
.9 Hz, 2H). LC-MS: m/z 394 (M+H) .
1
20 °C under N for 5 h. Upon completion, the reaction mixture was
2
6
-(But-3-en-1-yl)-4-(3-(difluoromethoxy)-5-(morpholine-4-
carbonyl)phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one
11). To a solution of 3-bromo-5-hydroxybenzoic acid (2.00 g, 9.2
concentrated in vacuo. The residue was dissolved in ethyl acetate (60
mL) and washed with water (2 × 30 mL). The separated organic layer
was concentrated under reduced pressure. The crude product was
purified by flash chromatography (1:1 petroleum ether/ethyl acetate)
to give (3-(difluoromethoxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2 yl)phenyl)(morpholino)methanone (300 mg, 66% yield) as a
(
mmol) in DMF (20 mL) were added HATU (3.89 g, 10.2 mmol),
DIPEA (1.32 g, 10.2 mmol), and morpholine (887 mg, 10.2 mmol).
The reaction was stirred at ambient temperature for 10 h and then
poured into water (40 mL) and extracted with ethyl acetate (3 × 50
mL). The combined organic layers were concentrated in vacuo. The
residue was purified by flash chromatography (1:2 petroleum ether/
ethyl acetate) to give (3-bromo-5-hydroxyphenyl)(morpholino)-
+
yellow oil. LC-MS: m/z 384.2 (M+H) .
6
-(But-3-en-1-yl)-4-(3-(difluoromethoxy)-5-(morpholine-4-
carbonyl)phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (11)
was then prepared in a similar procedure as shown for compound 3
except (3-(difluoromethoxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
methanone (900 mg, 31% yield) as a white solid. LC-MS: m/z
+
1
2
88.1 (M+H) .
lan-2-yl)phenyl) morpholine methanone was used instead. H NMR
I
DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
400 MHz, DMSO-d ) δ 12.18 (s, 1 H), 7.55−7.36 (m, 3H), 7.18−
6-(But-3-en-1-yl)-4-(4-methyl-3-(morpholine-4-carbonyl)-
phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (15).
Compound 15 was prepared in a similar manner as shown for
compound 12 except methyl 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-
6
7
4
2
.15 (m, 2H), 6.42 (s, 1H), 5.87−5.80 (m, 1H), 5.07−4.99 (m, 2H),
.11−4.08 (m, 2H), 3.63−3.53 (m, 4H), 3.36−3.26 (m, 4H), 2.48−
+
.46 (m, 2H). LC-MS: m/z 444 (M+H) .
3
-(6-(But-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]-
dioxaborolan-2-yl)benzoate was used instead.
1
pyridin-4-yl)-5-(morpholine-4-carbonyl)benzonitrile (12). To a
solution of 3,5-dibromobenzoic acid (2.79 g, 10 mmol) in DMF (30
mL) were added HATU (3.80 g, 10 mmol), DIPEA (1.32 g, 10.2
mmol), and morpholine (870 mg, 10.2 mmol). The reaction was
stirred at ambient temperature for 10 h and then poured into water
H NMR (400 MHz, DMSO-d ) δ 12.10 (s, 1H), 7.54 (dd, J = 8.0,
6
1.9 Hz, 1H), 7.45−7.21 (m, 4H), 6.39 (d, J = 2.8 Hz, 1H), 5.86 (ddt,
J = 17.0, 10.2, 6.7 Hz, 1H), 5.15−4.93 (m, 2H), 4.11 (t, J = 7.2 Hz,
2H), 3.59 (d, J = 50.2 Hz, 6H), 3.21 (s, 2H), 2.47 (d, J = 7.3 Hz, 2H),
+
2.27 (s, 3H). LC-MS: m/z 392 (M+H) .
(
40 mL) and extracted with ethyl acetate (3 × 150 mL). The
6-(But-3-en-1-yl)-4-(3,4-difluoro-5-(morpholine-4-
carbonyl)phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one
(16). Compound 16 was prepared in the same manner as shown for
combined organic layers were concentrated in vacuo. The residue was
purified by flash chromatography (1:2 petroleum ether/ethyl acetate)
to give (3,5-dibromophenyl)(morpholino)methanone (1.7 g, 49%
compound 12 except (3,4-difluoro-5-methoxycarbonyl-phenyl)-
+
1
yield) as a white solid. LC-MS: m/z 348.1, 350.1 (M+H) .
boronic acid was used instead. H NMR (400 MHz, DMSO-d
6
) δ
To a solution of (3,5-dibromophenyl)(morpholino)methanone
2.8 g, 7.3 mmol) in NMP (50 mL) was added CuCN (650 mg, 7.3
12.18 (s, 1H), 7.72 (ddd, J = 11.7, 7.5, 2.2 Hz, 1H), 7.48 (s, 1H),
7.45−7.36 (m, 2H), 6.45 (d, J = 2.8 Hz, 1H), 5.86 (ddt, J = 17.0, 10.2,
6.7 Hz, 1H), 5.14−4.99 (m, 2H), 4.10 (t, J = 7.3 Hz, 2H), 3.68−3.50
(m, 4H), 3.57 (t, J = 4.8 Hz, 2H), 3.35 (t, J = 4.7 Hz, 2H), 2.50−2.44
(
mmol). The reaction was heated at 175 °C for 2 h. After the mixture
was cooled to ambient temperature, it was poured into concentrated
ammonium hydroxide (200 mL), and the resulting mixture was
extracted with ethyl acetate (3 × 150 mL). The combined organic
layers were concentrated in vacuo. The residue was purified by flash
chromatography (1:1 petroleum ether/ethyl acetate) to give 3-bromo-
+
(m, 2H). LC-MS: m/z 414 (M+H) .
6-(But-3-en-1-yl)-4-(5-(morpholine-4-carbonyl)pyridin-3-
yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (17). Com-
pound 17 was prepared in the same manner as shown for compound
3
except [5-(morpholine-4-carbonyl)-3-pyridyl]boronic acid was used
5
-(morpholine-4-carbonyl)benzonitrile (1.0 g, 47% yield) as a yellow
1
+
instead. H NMR (400 MHz, DMSO-d ) δ 12.19 (s, 1H), 8.89 (d, J =
solid. LC-MS: m/z 295.1, 297.1 (M+H) .
6
A mixture of 3-bromo-5-(morpholine-4-carbonyl)benzonitrile
1.47 g, 5.0 mmol), bis(pinacolato)diboron (1.52 g, 6.0 mmol),
2.3 Hz, 1H), 8.58 (d, J = 1.9 Hz, 1H), 8.01 (t, J = 2.1 Hz, 1H), 7.56
(s, 1H), 7.40 (d, J = 2.8 Hz, 1H), 6.46 (d, J = 2.8 Hz, 1H), 5.87 (ddt,
J = 17.0, 10.2, 6.7 Hz, 1H), 5.18−4.95 (m, 2H), 4.13 (t, J = 7.2 Hz,
(
potassium acetate (588 mg, 6.0 mmol), and Pd(dppf)Cl (730 mg, 1.0
2
+
mmol) in dioxane (100 mL) was heated at 120 °C under N for 3 h.
2H), 3.65 (m, 8H), 2.47−2.43 (m, 2H). LC-MS: m/z 379 (M+H) .
2
Upon completion, the reaction mixture was concentrated in vacuo.
The residue was purified by flash chromatography (1:1 petroleum
ether/ethyl acetate) to give crude 3-(morpholine-4-carbonyl)-5-
6-(But-3-en-1-yl)-4-(6-methyl-5-(morpholine-4-carbonyl)-
pyridin-3-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (18).
Compound 18 was prepared in the same manner as shown for
compound 12 except ethyl 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (600 mg,
1
dioxaborolan-2-yl)pyridine-3-carboxylate was used instead. H NMR
3
5% yield) as a yellow oil.
A mixture of 4-bromo-6-(but-3-en-1-yl)-1,6-dihydro-7H-pyrrolo-
(
(
5
=
3
400 MHz, DMSO-d ) δ 12.17 (s, 1H), 8.74 (t, J = 1.6 Hz, 1H), 7.81
6
d, J = 1.9 Hz, 1H), 7.58−7.25 (m, 2H), 6.52−6.27 (m, 1H), 5.98−
[
2,3-c]pyridin-7-one (266 mg, 1.0 mmol), methyl 2-methyl-5-(4,4,5,5-
.71 (m, 1H), 5.23−4.93 (m, 2H), 4.11 (t, J = 7.3 Hz, 2H), 3.62 (d, J
tetramethyl-1,3,2-dioxaborolan-2-yl) benzoate (342 mg, 1.0 mmol),
56.5 Hz, 6H), 3.27−3.18 (m, 2H), 2.50−2.48 (m, 2H), 2.46 (s,
cesium carbonate (325 mg, 1.0 mmol), and Pd(dppf)Cl (50 mg, 0.06
2
+
H). LC-MS: m/z 393 (M+H) .
mmol) in dioxane (20 mL) and water (2 mL) was heated at reflux
temperature for 4 h under nitrogen atmosphere. The mixture was then
filtered through Celite, using ethyl acetate (20 mL) to rinse the cake.
The filtrate was concentrated in vacuo. The residue was purified by
preparative HPLC (35−65% ACN/0.1% NH HCO in water) to give
6
-(But-3-en-1-yl)-4-(6-methoxy-5-(morpholine-4-carbonyl)-
pyridin-3-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (19).
Compound 19 was prepared in the same manner as shown for
compound 12 except methyl 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-
4
3
1
dioxaborolan-2-yl)nicotinate was used instead. H NMR (400 MHz,
3
-(6-(but-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-
DMSO-d ) δ 12.14 (s, 1H), 8.46 (d, J = 2.5 Hz, 1H), 7.89 (d, J = 2.5
yl)-5-(morpholine-4-carbonyl)benzonitrile (32 mg, 8% yield) as a
6
1
Hz, 1H), 7.49−7.30 (m, 2H), 6.40 (d, J = 2.8 Hz, 1H), 5.97−5.78 (m,
white solid. H NMR (400 MHz, DMSO-d ) δ 12.21 (s, 1H), 8.09 (s,
6
1
H), 5.18−4.95 (m, 2H), 4.10 (t, J = 7.3 Hz, 2H), 3.96 (s, 3H), 3.65
1
1
3
H), 7.90 (s, 1H), 7.83 (s, 1H), 7.58 (s, 1H), 7.36 (s, 1H), 6.46 (s,
(s, 4H), 3.56 (t, J = 4.8 Hz, 2H), 3.23 (t, J = 4.7 Hz, 2H), 2.49−2.44
(m, 2H). LC-MS: m/z 409 (M+H) .
H), 5.87−5.70 (m, 1H), 5.08−4.98 (m, 2H), 4.11−4.06 (m, 2H),
+
.63−3.53 (m, 4H), 3.36−3.26 (m, 4H), 2.48−2.46 (m, 2H). LC-MS:
+
6-(But-3-en-1-yl)-4-(6-ethoxy-5-(morpholine-4-carbonyl)-
m/z 403.3 (M+H) .
pyridin-3-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (20).
A mixture of morpholine (1.11 g, 12.69 mmol), 5-bromo-2-
chloronicotinic acid (2.0 g, 8.46 mmol), 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro-
phosphate (3.37 g, 8.88 mmol), and triethylamine (2.36 mL, 16.9
mmol) in DMF (20 mL) was stirred at 18 °C for 2 h. The resulting
mixture was poured into water (100 mL) and extracted with ethyl
acetate (3 × 50 mL). The combined organic layers were dried over
sodium sulfate and concentrated under reduced pressure to give crude
6
-(But-3-en-1-yl)-4-(4-fluoro-3-(morpholine-4-carbonyl)-
phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (13).
Compound 13 was prepared in the same manner as shown for
compound 3 except (4-fluoro-3-(morpholine-4-carbonyl)phenyl)-
boronic acid was used instead. H NMR (400 MHz, DMSO-d ) δ
2.14 (s, 1H), 7.71 (m, J = 8.6, 5.1, 2.4 Hz, 1H), 7.58 (m, J = 6.4, 2.4
1
6
1
Hz, 1H), 7.47−7.33 (m, 3H), 6.41 (d, J = 2.9 Hz, 1H), 5.95−5.78 (m,
1
3
(
H), 5.15−4.96 (m, 2H), 4.20−4.02 (m, 2H), 3.67−3.60 (m, 4H),
.56 (m, J = 5.1 Hz, 4H), 2.49 (m, J = 4.7 Hz, 2H). LC-MS: m/z 396
+
(
5-bromo-2-chloropyridin-3-yl) (morpholino)methanone (2.2 g,
M+H) .
6
+
-(But-3-en-1-yl)-4-(4-chloro-3-(morpholine-4-carbonyl)-
85%) as a brown solid. LC-MS: m/z 306.9 (M+H) .
phenyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (14).
Compound 14 was prepared in the same manner as shown for
To a freshly prepared sodium ethanolate solution, in which sodium
(56 mg, 1.64 mmol) was added carefully to ethanol (10 mL) and
stirred until no solid remained, was added (5-bromo-2-chloropyridin-
3-yl)(morpholino)methanone (500 mg, 1.64 mmol). The mixture was
heated at 90 °C for 2 h and then concentrated under reduced
pressure. The residue was diluted with water (20 mL) and extracted
with ethyl acetate (3 × 10 mL). The combined organics were dried
over sodium sulfate and concentrated under reduced pressure to give
compound 3 except (4-chloro-3-(morpholine-4-carbonyl)phenyl)-
boronic acid was used instead. H NMR (400 MHz, DMSO-d ) δ
1.92 (s, 1H), 7.65 (dd, J = 8.5, 2.2 Hz, 1H), 7.62−7.51 (m, 3H),
.42 (s, 1H), 6.15 (dd, J = 1.9, 1.0 Hz, 1H), 5.85 (ddt, J = 17.0, 10.2,
.7 Hz, 1H), 5.19−4.96 (m, 2H), 4.09 (t, J = 7.3 Hz, 2H), 3.82−3.21
m, 8H), 2.47 (t, J = 6.6 Hz, 2H). LC-MS: m/z 426 (M+H) .
1
6
1
7
6
+
(
J
DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
crude (5-bromo-2-ethoxypyridin-3-yl) (morpholino)methanone (380
mg, 74%) as a brown solid. LC-MS: m/z 314.9 (M+H) .
A mixture of (5-bromo-2-ethoxypyridin-3-yl)(morpholino)-
methanone (380 mg, 1.21 mmol), bis(pinanolato)diboron (460 mg,
mL) was added trifluoroacetic acid (10 mL). The mixture was stirred
at ambient temperature for 3 h and then concentrated under reduced
pressure to give 6-(but-3-en-1-yl)-4-(4-(morpholine-4-carbonyl)-1H-
benzo[d]imidazol-6-yl)-1-tosyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-one
+
+
1
.78 mmol), Pd(dppf)Cl (38 mg), and potassium acetate (236 mg,
(400 mg, 98% yield) as a yellow oil. LC-MS: m/z 572.1 (M+H) .
2
2
.39 mmol) in dioxane (10 mL) was heated at 120 °C under
To a stirred and cooled (0 °C) solution of 6-(but-3-en-1-yl)-4-(4-
(morpholine-4-carbonyl)-1H-benzo[d]imidazol-6-yl)-1-tosyl-1H-
pyrrolo[2,3-c]pyridin-7(6H)-one (500 mg, 0.87 mmol) in DMF (10
mL) was slowly added sodium hydride (60%, 42 mg, 0.92 mmol).
After addition, the mixture was stirred at 0 °C for 1 h, and then
iodomethane (149 mg, 0.92 mmol) was added. The resulting mixture
was stirred at ambient temperature for 2 h. Upon completion, the
reaction was quenched by the addition of saturated aqueous
ammonium chloride (10 mL) and then extracted with ethyl acetate
(3 × 20 mL). The combined organic layers were concentrated in
vacuo. The residue was purified by silica-gel chromatography (1:1
hexanes/ethyl acetate) to give the mixture of the methylated 6-but-3-
enyl-4-[3-methyl-7-(morpholine-4-carbonyl)benzimidazol-5-yl]-1-
tosyl-1H-pyrrolo[2,3-c]pyridin-7-one and 6-but-3-enyl-4-[1-methyl-7-
(morpholine-4-carbonyl)benzimidazol-5-yl]-1-tosyl-1H-pyrrolo[2,3-
c]pyridin-7-one regioisomers (250 mg, 49% yield) as a brown solid.
To a solution of the above regioisomers (250 mg, 0.43 mmol) in
microwave irradiation for 1 h. The mixture was concentrated under
reduced pressure, and the residue was purified by preparative TLC
(
(
5% methanol in dichloromethane, R = 0.5) to afford (6-ethoxy-5-
morpholine-4-carbonyl)pyridin-3-yl)boronic acid (220 mg, 65%) as
f
+
brown solid. LC-MS: m/z 280.9 (M+H) .
A mixture of 4-bromo-6-(but-3-en-1-yl)-1-tosyl-1H-pyrrolo[2,3-
c]pyridin-7 (6H)-one (28, 220 mg, 0.52 mmol), (6-ethoxy-5-
(
morpholine-4-carbonyl)pyridin-3-yl) boronic acid (220 mg, 0.78
mmol), Pd(dppf)Cl (10 mg), and cesium carbonate (340 mg, 1.04
2
mmol) in dioxane/water (6:1 mL) was heated at 110 °C for 1 h under
microwave conditions. The resulting mixture was concentrated under
reduced pressure, and the residue was purified by preparative TLC
(
1
50% ethyl acetate in petroleum ether, R = 0.2) to afford 6-(but-3-en-
-yl)-4-(6-ethoxy-5-(morpholine-4-carbonyl)pyridin-3-yl)-1-tosyl-1H-
f
pyrrolo[2,3-c]pyridin-7(6H)-one (120 mg, 41%) as brown oil. LC-
MS: m/z 423 (M+H) .
+
A mixture of 6-but-3-enyl-4-[6-ethoxy-5-(morpholine-4-carbonyl)-
-pyridyl]-1-(p-tolylsulfonyl)pyrrolo[2,3-c]pyridin-7-one (120 mg,
.21 mmol) and 10% aqueous sodium hydroxide (0.17 mL, 0.21
dioxane/H O (10 mL, 1:1) was added sodium hydroxide (34 mg,
2
3
0
0.86 mmol). The reaction was heated at 80 °C for 3 h and then
concentrated in vacuo and partitioned between dichloromethane (40
mL) and water (30 mL). The separated organic layer was
concentrated under reduced pressure, and the residue was purified
mmol) in methanol (15 mL) was heated at 90 °C for 1 h and then
concentrated to dryness under reduced pressure. The residue was
diluted with water (20 mL). The crude solid product was collected by
filtration and purified by preparative HPLC (32−52% ACN/0.1%
by reverse-phase chromatography (18−48% ACN/0.1% NH OH in
4
water), resulting in the title compounds as white solids. Compound
1
NH OH in water) to give 6-(but-3-en-1-yl)-4-(6-ethoxy-5-(morpho-
22 (11 mg, 6% yield). H NMR (400 MHz, CD OD) δ 9.54 (s, 1H),
4
3
line-4-carbonyl)pyridin-3-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-
8.14 (s, 1H), 7.87 (s, 1H), 7.48 (s, 2H), 6.55 (d, J = 2.8 Hz, 1H),
5.96−5.90 (m, 1H), 5.13−5.06 (m, 2H), 4.27−4.24 (m, 2H), 4.14 (s,
3H), 4.04−3.54 (m, 8H), 2.64−2.59 (m, 2H). LC-MS: m/z 432 (M
1
one (30 mg, 34%) as a white solid. H NMR (400 MHz, DMSO-d ) δ
6
1
1
4
2
2.16 (s, 1H), 8.44 (s, 1H), 7.88 (s, 1H), 7.43−7.37 (m, 2H), 6.41 (s,
H), 5.91−5.80 (m, 1H), 5.09−5.01 (m, 2H), 4.50−4.30 (m, 2H),
.12−4.08 (m, 2H), 3.78−3.55 (m, 4H), 3.33−3.23 (m, 4H), 2.50−
+
1
+H) . Compound 23 (6.3 mg, 3% yield). H NMR (400 MHz,
CD OD) δ 9.59 (s, 1H), 8.26 (s, 1H), 7.95 (s, 1H), 7.53−7.49 (m,
3
+
.45 (m, 2H), 1.37−1.33 (m, 3H). LC-MS: m/z 578 (M+H) .
2H), 7.62 (d, J = 2.8 Hz, 1 H), 5.98−5.89 (m, 1H), 5.13−5.06 (m,
2H), 4.29−4.26 (m, 5H), 4.02−3.46 (m, 8H), 2.66−2.61 (m, 2H).
6
-But-3-enyl-4-[7-(morpholine-4-carbonyl)-3H-benzimida-
+
zol-5-yl]-1H-pyrrolo[2,3-c]pyridin-7-one (21). To a solution of 6-
but-3-en-1-yl)-4-(4-(morpholine-4-carbonyl)-1-((2-(trimethylsilyl)
LC-MS: m/z 432 (M+H) .
(
6-(But-3-en-1-yl)-4-(1-ethyl-4-(morpholine-4-carbonyl)-1H-
benzo[d]imidazol-6-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-
7-one and 6-(But-3-en-1-yl)-4-(1-ethyl-7-(morpholine-4-car-
bonyl)-1H-benzo[d]imidazol-5-yl)-1,6-dihydro-7H-pyrrolo[2,3-
c]pyridin-7-one (24 and 25). Compounds 24 and 25 were
prepared in the same manner as shown for compounds 22 and 23
ethoxy)methyl)-1H-benzo[d]imidazol-6-yl)-1-tosyl-1H-pyrrolo[2,3-
c]pyridin-7(6H)-one 37 (500 mg, 0.71 mmol) in dioxane/H O (10
2
mL, 1:1) was added sodium hydroxide (57 mg, 1.42 mmol). The
reaction mixture was heated at 80 °C for 3 h, at which time LC-MS
indicated the reaction had gone to completion. The mixture was
concentrated under reduced pressure. The residue was diluted with
ethyl acetate (30 mL), washed with water (2 × 20 mL), and
concentrated under reduced pressure to give the crude 6-(but-3-en-1-
yl)-4-(4-(morpholine-4-carbonyl)-1-((2-(trimethylsilyl)ethoxy)-
methyl)-1H-benzo[d]imidazol-6-yl)-1H-pyrrolo[2,3-c]pyridin-7(6H)-
one (350 mg, 90% yield).
To a solution of the above crude in dichloromethane (15 mL) was
added trifluoroacetic acid (15 mL). After addition, the reaction
mixture was stirred at ambient temperature for 3 h, at which time LC-
MS indicated that the reaction had gone to completion. The reaction
mixture was concentrated under reduced pressure. The crude was
purified by reverse-phase chromatography (13−33% ACN/0.1% HCl
in water) to give 6-but-3-enyl-4-[7-(morpholine-4-carbonyl)-3H-
1
with iodoethane used instead. Compound 24 (71 mg, 12% yield). H
NMR (400 MHz, CD OD) δ 8.34 (s, 1H), 7.98 (s, 1 H), 7.49 (s,
3
1
6
4
2
H), 7.42 (d, J = 2.8 Hz, 1H), 7.31 (s, 1H), 6.51 (d, J = 2.8 Hz, 1H),
.97−5.88−5.79 (m, 1H), 5.12−5.04 (m, 2H), 4.41−4.39 (m, 2H),
.24−4.20 (m, 2H), 3.92−3.81 (m, 4H), 3.69−3.31 (m, 4H), 2.62−
+
.56 (m, 2H), 1.49 (t, J = 7.2 Hz, 3H). LC-MS: m/z 446 (M+H) .
1
Compound 25 (105 mg, 26% yield). H NMR (400 MHz, CD OD) δ
3
8
6
2
4
.32 (s, 1H), 7.87 (s, 1H), 7.54 (s, 1H), 7.40 (s, 1H), 7.35 (s, 1H),
.58 (s, 1H), 5.93−5.87 (m, 1H), 5.10−5.02 (m, 2H), 4.43−4.38 (m,
H), 4.21 (t, J = 7.2 Hz, 2H), 3.88−3.81 (m, 4H), 3.62−3.35 (m,
H), 2.61−2.56 (m, 2H), 1.55 (t, J = 7.2 Hz, 3H). LC-MS: m/z 446
+
(M+H) .
6
-But-3-enyl-4-[6-(morpholine-4-carbonyl)-1H-benzimida-
zol-4-yl]-1H-pyrrolo[2,3-c]pyridin-7-one (26). To a solution of
methyl 4-(6-(but-3-en-1-yl)-7-oxo-1-tosyl-6,7-dihydro-1H-pyrrolo
benzimidazol-5-yl]-1H-pyrrolo[2,3-c]pyridin-7-one (11 mg, 5%
1
yield). H NMR (400 MHz, CD OD) δ 9.58 (s, 1H), 8.16 (s, 1H),
3
[
2,3-c]pyridin-4-yl)-1H-benzo[d]imidazole-6-carboxylate (42, 400
7
.91 (s, 1H), 7.48 (s, 2 H), 6.57 (s, 1H), 5.97−5.88 (m, 1H), 5.09 (t,
mg, 0.77 mmol) in methanol (10 mL) was added a solution of
sodium hydroxide (1 N in water, 2.0 mL, 2.0 mmol) at ambient
temperature. The resulting mixture was heated at 100 °C for 2 h.
After cooling to ambient temperature, the mixture was adjusted to pH
J = 10.4 Hz, 2H), 4.26 (t, J = 7.2 Hz, 2 H), 4.02−3.48 (m, 8 H),
+
2
.65−2.57 (m, 2 H). LC-MS: m/z 418 (M+H) .
-(But-3-en-1-yl)-4-(1-methyl-4-(morpholine-4-carbonyl)-
H-benzo[d]imidazol-6-yl)-1,6-dihydro-7H-pyrrolo[2,3-c]-
6
1
3
−4 by addition of 1 N aqueous HCl. The resulting mixture was
pyridin-7-one and 6-(But-3-en-1-yl)-4-(1-methyl-7-(morpho-
line-4-carbonyl)-1H-benzo[d]imidazol-5-yl)-1,6-dihydro-7H-
pyrrolo[2,3-c]pyridin-7-one (22 and 23). To a solution of 6-(but-
extracted with ethyl acetate (3 × 30 mL). The combined organic
layers were concentrated under reduced pressure to give 4-(6-(but-3-
en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-1H-benzo-
3
-en-1-yl)-4-(4-(morpholine-4-carbonyl)-1-((2-(trimethylsilyl)
ethoxy)methyl)-1H-benzo[d]imidazol-6-yl)-1-tosyl-1H-pyrrolo[2,3-
c]pyridin-7(6H)-one (500 mg, 0.71 mmol) in dichloromethane (10
[d]imidazole-6-carboxylic acid (200 mg, 74% yield) as a white solid.
1
H NMR (400 MHz, DMSO-d ) δ 12.94 (br s, 1H), 12.16 (s, 1H),
6
K
DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
8
.84 (s, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 7.67 (s, 1H), 7.45 (d, J = 8.0
(morpholino)methanone (5.0 g, 76% yield) as a yellow solid. LC-
MS: m/z 332 (M+H) .
+
Hz, 0.5 H), 7.34 (s, 1H), 7.07 (d, J = 7.6 Hz, 0.5 H), 6.24 (s, 1H),
5
2
.87−5.80 (m, 1H), 5.08−4.97 (m, 2H), 4.13−4.09 (m, 2H), 2.50−
(2,3-Diamino-5-bromophenyl)(morpholino)methanone
+
.48 (m, 1H), 2.25 (s, 1H). LC-MS: m/z 418 (M+H) .
(33). To a solution of (2-amino-5-bromo-3-nitrophenyl)-
A mixture of 4-(6-(but-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo-
(morpholino)methanone (32, 5.0 g, 15.15 mmol) in EtOH/H
O
2
[
2,3-c]pyridine-4-yl)-1H-benzo[d]imidazole-6-carboxylic acid (100
(5:1, 100 mL) were added NH Cl (4.1 g, 75.73 mmol) and iron
4
powder (4.2 g, 75.73 mmol). The reaction mixture was heated at
reflux temperature for 15 h, at which time LC-MS indicated that the
reaction had gone to completion. The solid was removed by filtration,
and the filtrate was concentrated under reduced pressure. The residue
was dissolved in ethyl acetate (100 mL) and washed with brine (2 ×
mg, 0.28 mmol), morpholine (73 mg, 0.84 mmol), HATU (141
mg, 0.37 mmol), and DIPEA (72 mg, 0.56 mmol) in DMF (10 mL)
was heated at 60 °C for 8 h. The reaction mixture was concentrated in
vacuo, and the residue was dissolved in dichloromethane (50 mL),
washed with brine (2 × 20 mL), and concentrated under reduced
pressure. The crude product was purified by preparative HPLC(10−
40 mL). The separated organic layer was concentrated to give (2,3-
diamino-5-bromophenyl)(morpholino)methanone (4.2 g, 92% yield)
4
0% ACN/0.1% HCl in water) to give 6-but-3-enyl-4-[6-(morpho-
+
as a yellow oil. LC-MS: m/z 302.0 (M+H) .
line-4-carbonyl)-1H-benzimidazol-4-yl]-1H-pyrrolo[2,3-c]pyridin-7-
1
(6-Bromo-1H-benzo[d]imidazol-4-yl)(morpholino)-
methanone (34). To a solution of (2,3-diamino-5-bromophenyl)-
one as a white solid (25 mg, 21% yield). H NMR (400 MHz,
CD OD) δ 9.56 (s, 1H), 7.97 (d, J = 4.0 Hz, 1H), 7.78 (s, 1H), 7.58
3
(
morpholino)methanone (33, 4.2 g, 12.72 mmol) in DMF (40 mL)
(
(
s, 1H), 7.45 (d, J = 2.8 Hz, 1 H), 6.28 (d, J = 2.8 Hz, 1H), 5.94−5.88
were added triethoxymethane (2.8 g, 19.08 mmol) and 4-
methylbenzenesulfonic acid (220 mg, 1.27 mmol). The reaction
mixture was stirred at ambient temperature for 15 h, at which time
LC-MS indicated that the reaction had gone to completion. The
solvent was evaporated under reduced pressure, and the residue was
diluted with water (40 mL). The mixture was extracted with ethyl
acetate (3 × 50 mL). The combined organic extracts were
concentrated under reduced pressure. The crude product was purified
by silica-gel chromatography (10:1 DCM/MeOH) to give (6-bromo-
m, 1H), 5.15−5.05 (m, 2H), 4.27 (t, J = 7.2 Hz, 2H), 3.93−3.47 (m,
+
8
H), 2.65−2.59 (m, 2H). LC-MS: m/z 418 (M+H) .
6
-But-3-enyl-4-[1-methyl-6-(morpholine-4-carbonyl)-
benzimidazol-4-yl]-1H-pyrrolo[2,3-c]pyridin-7-one (27, GNE-
71). To a solution of methyl 4-(6-(but-3-en-1-yl)-7-oxo-1-tosyl-6,7-
3
dihydro-1H-pyrrolo [2,3-c]pyridin-4-yl)-1-methyl-1H-benzo[d]-
imidazole-6-carboxylate (43, 350 mg, 0.66 mmol) in methanol/H O
2
(
15:3 mL) was added sodium hydroxide (130 mg, 3.3 mmol). The
reaction was heated at 80 °C for 16 h and then concentrated in vacuo.
1
H-benzo[d]imidazol-4-yl)(morpholino)methanone (3.0 g, 76%
The residue was diluted with H O (8 mL), and the solution was
2
+
yield) as a brown solid. LC-MS: m/z 311.9 (M+H) .
adjusted to pH 5−6 by the addition of 2 N aqueous HCl. The
resulting precipitate was collected by filtration, washed with water and
dried under reduced pressure to give 4-(6-(but-3-en-1-yl)-7-oxo-6,7-
dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-1-methyl-1H-benzo[d]-
imidazole-6-carboxylic acid (220 mg, 92% yield) as a yellow solid. LC-
(
6-Bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]-
imidazol-4-yl)(morpholino)methanone (35). To a solution of (6-
bromo-1H-benzo[d]imidazol-4-yl)(morpholino)methanone (34, 3.0
g, 9.67 mmol) in DMF (50 mL) was slowly added sodium hydride
(60%, 464 mg, 11.61 mmol) at 0 °C. After addition, the mixture was
stirred at 0 °C for 1 h, and then (2-(chloromethoxy)ethyl)-
trimethylsilane (1.9 g, 11.61 mmol) was added dropwise. The
resulting mixture was stirred at ambient temperature for another 2 h,
at which time LC-MS indicated that the reaction had gone to
completion. The reaction was quenched by the addition of saturated
aqueous ammonium chloride (50 mL) and then extracted with ethyl
acetate (3 × 50 mL). The combined organic extracts were
concentrated under reduced pressure. The crude product was purified
by silica-gel chromatography (1:1 hexanes/ethyl acetate) to give (6-
bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-4-
yl)(morpholino)methanone (3.0 g, 70% yield) as a yellow solid. LC-
+
MS: m/z 362.9 (M+H) .
To a solution of 4-(6-(but-3-en-1-yl)-7-oxo-6,7-dihydro-1H-
pyrrolo[2,3-c]pyridin-4-yl)-1-methyl-1H-benzo[d]imidazole-6-carbox-
ylic acid (220 mg, 0.61 mmol) in DMF (5 mL) were added
triethylamine (120 mg, 1.2 mmol), HATU (460 mg, 1.2 mmol) and
morpholine (110 mg, 1.2 mmol). The reaction was stirred at ambient
temperature for 16 h and then concentrated in vacuo. The residue was
purified by reverse-phase chromatography (19% ACN/0.1% NH OH
4
in water) to give 6-but-3-enyl-4-[1-methyl-6-(morpholine-4-
carbonyl)benzimidazol-4-yl]-1H-pyrrolo[2,3-c]pyridin-7-one (86 mg,
1
3
3% yield) as a white solid. H NMR (400 MHz, DMSO-d ) δ 12.09
6
(
s, 1H), 8.33 (s, 1H), 7.84 (s, 1H), 7.64 (s, 1H), 7.46 (s, 1H), 7.35−
+
MS: m/z 441 (M+H) .
7
4
2
.34 (m, 1H), 6.37 (s, 1H), 5.93−5.84 (m, 1H), 5.12−5.07 (m, 2H),
Morpholino(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-
.15−4.11 (m, 2H), 4.08 (s, 3H), 3.63−3.57 (m, 8H), 2.46−2.44 (m,
+
13
H). LC-MS: m/z 432 (M+H) . C NMR (101 MHz, DMSO, 360
4
-yl)methanone (36). A mixture of (6-bromo-1-((2-
K) δ 170.30, 154.20, 146.14, 142.16, 135.61, 135.45, 130.52, 130.18,
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol −4-yl)-
morpholino)methanone (35, 3.0 g, 6.81 mmol), bis(pinanolato)-
1
1
29.45, 128.77, 127.17, 124.12, 120.22, 117.29, 111.54, 108.39,
(
03.39, 66.72, 47.38, 45.79, 34.01, 31.33. HRMS: calcd for
diboron (3.6 g, 13.62 mmol), potassium acetate (1.7 g, 17.64 mmol),
+
C H N O m/z (M+H) 432.2030, found 432.2029.
24
26
5
3
and Pd(dppf)Cl (0.5 g, 0.68 mmol) in dioxane (45 mL) was heated
2
(2-Amino-3-nitrophenyl)(morpholino)methanone (31). To a
at 110 °C under microwave irradiation for 45 min, at which time LC-
MS indicated that the reaction had gone to completion. The mixture
was concentrated under reduced pressure. The residue was dissolved
in ethyl acetate (80 mL), washed with water (2 × 50 mL), and
concentrated in vacuo to give morpholino(6-(4,4,5,5-tetramethyl-
solution of morpholine (2.4 g, 27.45 mmol) in DMF (80 mL) were
added 2-amino-3-nitrobenzoic acid (30, 5.0 g, 27.45 mmol), HATU
(
12.5 g, 32.94 mmol), and DIPEA (10 mL). The reaction was stirred
at ambient temperature for 15 h and then concentrated in vacuo. The
residue was diluted with ethyl acetate (100 mL) and washed with
water (2 × 50 mL). The organic solution was concentrated under
reduced pressure, and the residue was purified by silica-gel
chromatography (3:1 hexanes/ethyl acetate) to give (2-amino-3-
nitrophenyl)(morpholino)methanone (5.0 g, 73% yield) as a yellow
1
,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
benzo[d]imidazol-4-yl)methanone (1.35 g, 41% yield) as a brown oil.
LC-MS: m/z 405.9 (M+H) .
+
6
-(But-3-en-1-yl)-4-(4-(morpholine-4-carbonyl)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-6-yl)-1-
tosyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-one (37). A mixture of
morpholino(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-4-yl)methanone
(450 mg, 0.92 mmol), 4-bromo-6-(but-3-en-1-yl)-1-tosyl-1H-pyrrolo-
+
solid. LC-MS: m/z 252 (M+H) .
(
2-Amino-5-bromo-3-nitrophenyl)(morpholino)methanone
(
32). To a solution of (2-amino-3-nitrophenyl)(morpholino)-
methanone (31, 5.0 g, 19.90 mmol) in acetic acid (50 mL) was
added Br (3.82 g, 23.88 mmol). The reaction was stirred at 0 °C for
[2,3-c]pyridin-7(6H)-one (36, 389 mg, 0.92 mmol), Pd(dppf)Cl (66
mg, 0.09 mmol), and cesium carbonate (601 mg, 1.85 mmol) in
2
2
3
0 min. The mixture was poured into ice-cold water (30 mL). The
resulting precipitate was collected by filtration and dried under
reduced pressure to give (2-amino-5-bromo-3-nitrophenyl)-
dioxane/H O (10 mL, 4:1) was heated at 110 °C under microwave
2
irradiation for 45 min, at which time LC-MS indicated the reaction
L
DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
had gone to completion. The mixture was concentrated under
reduced pressure. The residue was dissolved in ethyl acetate (30 mL),
washed with water (2 × 20 mL), and concentrated under reduced
pressure. The residue was purified by silica-gel chromatography (2:1
hexanes/ethyl acetate) to give 6-(but-3-en-1-yl)-4-(4-(morpholine-4-
carbonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]-
Synthesis of TAF1(2)-Tracer Probe N-(2-(2-((6-(6-ROX)-
amidohexyl)oxy)ethoxy)ethyl)-5-(6-(but-3-en-1-yl)-7-oxo-6,7-
dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-2-methoxynicotina-
mide. A mixture of 4-bromo-6-(but-3-en-1-yl)-1-tosyl-1H-pyrrolo-
[2,3-c]pyridin-7(6H)-one 28 (500 mg, 1.2 mmol), methyl 2-methoxy-
5
1
-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate (370 mg,
.2 mmol), Pd(dppf)Cl2 (90 mg, 0.1 mmol), and cesium carbonate
imidazol-6-yl)-1-tosyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-one (500 mg,
+
(785 mg, 2.0 mmol) in 1,4-dioxane (15 mL)/water (3 mL) was
heated at 110 °C for 2 h under nitrogen atmosphere. After the
mixture was cooled, it was extracted with ethyl acetate (20 mL × 2).
The combined organic layers were washed with brine, dried over
sodium sulfate, and concentrated under reduced pressure. The residue
was purified by flash column chromatography (0−20% ethyl acetate in
petroleum ether) to give methyl 5-(6-(but-3-en-1-yl)-7-oxo-1-tosyl-
6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-2-methoxynicotinate (370
7
7% yield) as a yellow solid. LC-MS: m/z 703 (M+H) .
Methyl 3,4-Diamino-5-bromobenzoate (39). A mixture of
methyl 4-amino-3-bromo-5-nitrobenzoate (38, 1.0 g, 3.6 mmol) and
SnCl ·2H O (1.6 g, 7.3 mmol) in ethyl acetate (30 mL) was heated at
2
2
9
0 °C for 18 h. Upon completion, the reaction was cooled, diluted
with water (30 mL), and extracted with ethyl acetate (3 × 30 mL).
The combined organic layers were concentrated under reduced
pressure to give methyl 3,4-diamino-5-bromobenzoate (850 mg, 95%
1
+
yield) as a yellow solid. H NMR (400 MHz, CDCl ) δ 7.74 (d, J =
mg, 62%) as a yellow oil. LC-MS: m/z = 508 (M+H)
3
1
2
.6 Hz, 1H), 7.35 (s, 1H), 4.25 (br s, 2H), 3.86 (s, 3H), 3.50 (br s,
To a solution of methyl 5-(6-(but-3-en-1-yl)-7-oxo-1-tosyl-6,7-
dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-2-methoxynicotinate (370
mg, 0.7 mmol) in 1,4-dioxane (3 mL) and water (1 mL) was
added sodium hydroxide (146 mg, 3.6 mmol). The reaction mixture
was stirred at 75 °C for 3 h and then concentrated under reduced
pressure. The residue was adjusted to pH 5 by addition of
hydrochloric acid (2 N). The resulting mixture was extracted with
ethyl acetate (3 × 20 mL). The combined organic layers were washed
with water (20 mL) and brine (20 mL), dried over anhydrous sodium
sulfate, and concentrated under reduced pressure to afford crude 5-(6-
(but-3-en-1-yl)-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-2-
methoxynicotinic acid as a yellow solid (220 mg, 88%). LC-MS: m/z
H).
Methyl 4-Bromo-1H-benzo[d]imidazole-6-carboxylate (40).
To a solution of methyl 3,4-diamino-5-bromobenzoate (850 mg, 3.5
mmol) in THF (30 mL) were added triethoxymethane (1.04 g, 7.0
mmol) and TsOH·H O (66 mg, 0.35 mmol). The resulting mixture
was stirred at ambient temperature for 5 h and then concentrated in
vacuo. The residue was diluted with water (30 mL) and extracted with
ethyl acetate (3 × 30 mL). The combined organic layers were
concentrated under reduced pressure to give methyl 4-bromo-1H-
2
benzo[d]imidazole-6-carboxylate (820 mg, 93% yield) as a pale-white
solid. H NMR (400 MHz, CDCl ) δ 8.40 (s, 1H), 8.22 (s, 2H), 3.97
1
3
+
(
s, 3H).
= 340 (M+H)
(
6-(Methoxycarbonyl)-1H-benzo[d]imidazol-4-yl)boronic
To a cooled solution of tert-butyl N-[2-[2-(6-aminohexoxy)-
ethoxy]ethyl]carbamate (882 mg, 2.9 mmol) in 1,4-dioxane (2.5
mL) at 0 °C was added a solution of sodium bicarbonate (730 mg, 8.7
mmol) in water (5 mL); this was followed by the addition of 9H-
fluoren-9-ylmethyl carbonochloridate (1130 mg, 4.4 mmol) in 1,4-
dioxane (2.5 mL). The reaction mixture was stirred at room
temperature for 18 h, and the resulting mixture then was poured
into water (20 mL) and extracted with ethyl acetate (2 × 20 mL).
The combined organic layers were washed with brine, dried over
sodium sulfate, and concentrated under reduced pressure. The residue
was purified by flash column chromatography (0−25% ethyl acetate in
petroleum ether) to give 9H-fluoren-9-ylmethyl N-[6-[2-[2-(tert-
Acid (41). A mixture of methyl 4-bromo-1H-benzo[d]imidazole-6-
carboxylate (820 mg, 3.21 mmol), bis(pinanolato)diboron (1.2 g, 4.5
mmol), Pd(dppf)Cl (230 mg, 0.32 mmol), and potassium acetate
2
(
630 mg, 6.4 mmol) in dioxane (30 mL) was heated at 120 °C under
N atmosphere for 16 h. Upon completion, the mixture was cooled,
2
filtered through a Celite pad, and rinsed with dioxane (20 mL). The
filtrate was concentrated in vacuo. The residue was diluted with water
(
50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined
organic layers were concentrated under reduced pressure to give (6-
methoxycarbonyl)-1H-benzo[d]imidazol-4-yl)boronic acid as a
brown solid (650 mg, 92% crude yield). LC-MS: m/z 221 (M+H) .
Methyl 4-(6-(But-3-en-1-yl)-7-oxo-1-tosyl-6,7-dihydro-1H-
pyrrolo[2,3-c]pyridin-4-yl)-1H-benzo[d]imidazole-6-carboxy-
late (42). A mixture of 4-bromo-6-(but-3-en-1-yl)-1-tosyl-1H-
pyrrolo[2,3-c]pyridin-7(6H)-one (28, 420 mg, 1.0 mmol), (6-
(
+
butoxycarbonylamino)ethoxy]ethoxy]hexyl]carbamate (262 mg,
1
1
7
(
3
7%) as a colorless oil. H NMR (400 MHz, CD3OD) δ 7.82−
.78 (m, 2H), 7.68−7.65 (m, 2H), 7.45−7.35 (m, 2H), 7.34−7.30
m, 2H), 4.62 (br s, 2H), 4.40−4.34 (m, 2H), 4.23−4.21 (m, 1H),
(
methoxycarbonyl)-1H-benzo[d]imidazol-4-yl)boronic acid (41, 330
mg, 1.5 mmol), cesium carbonate (650 mg, 2.0 mmol), and
Pd(dppf)Cl (73 mg, 0.10 mmol) in dioxane/H O (5:1, 30 mL)
.59−3.57 (m, 4H), 3.52−3.46 (m, 4H), 3.30−3.22 (m, 2H), 3.20−
.10 (m, 2H), 1.70−1.20 (m, 17H).
3
2
2
To a solution of 9H-fluoren-9-ylmethyl N-[6-[2-[2-(tert-
was heated at 100 °C under N for 3 h. The reaction mixture was
2
butoxycarbonylamino)ethoxy]ethoxy] hexyl]carbamate (262 mg, 0.5
mmol) in ethyl acetate (1 mL) was added hydrochloric acid (1.0 M in
ethyl acetate, 10 mL, 10.0 mmol). The mixture was stirred at 0 °C for
concentrated in vacuo, and the residue was diluted with water (30
mL) and extracted with ethyl acetate (3 × 30 mL). The combined
organic layers were concentrated under reduced pressure. The residue
was purified by silica-gel chromatography (99:1 to 16:1 DCM/
MeOH) to give methyl 4-(6-(but-3-en-1-yl)-7-oxo-1-tosyl-6,7-dihy-
dro-1H-pyrrolo[2,3-c]pyridin-4-yl)-1H-benzo[d]imidazole-6-carboxy-
1
h and concentrated under reduced pressure to give 9H-fluoren-9-
ylmethyl N-[6-[2-(2-aminoethoxy) ethoxy]hexyl]carbamate hydro-
chloride (230 mg, 100%) as a white solid. The crude product was
+
used in the next step without further purification. LC-MS: m/z = 427
late as a white solid (400 mg, 77% yield). LC-MS: m/z 516 (M+H) .
+
(M+H)
Methyl 4-(6-(But-3-en-1-yl)-7-oxo-1-tosyl-6,7-dihydro-1H-
pyrrolo[2,3-c]pyridin-4-yl)-1-methyl-1H-benzo[d]imidazole-6-
carboxylate (43). To a solution of methyl 4-(6-(but-3-en-1-yl)-7-
oxo-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)-1H-benzo[d]-
imidazole-6-carboxylate (42, 290 mg, 0.56 mmol) in DMF (4 mL)
were added potassium carbonate (160 mg, 1.1 mmol) and CH I (82
mg, 0.84 mmol). The reaction mixture was stirred at ambient
temperature for 6 h, at which time it was concentrated in vacuo. The
residue was diluted with H O (30 mL) and extracted with ethyl
acetate (3 × 30 mL). The combined organic layers were concentrated
to give methyl 4-(6-(but-3-en-1-yl)-7-oxo-1-tosyl-6,7-dihydro-1H-
pyrrolo[2,3-c]pyridin-4-yl)-1-methyl-1H-benzo[d]imidazole-6-carbox-
To a stirred solution of 9H-fluoren-9-ylmethyl N-[6-[2-(2-
aminoethoxy)ethoxy]hexyl]carbamate hydrochloride (220 mg, 0.48
mmol) and DIPEA (245 mg, 1.90 mmol) in DMF (10 mL) were
added 5-(6-but-3-enyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-2-me-
thoxy-pyridine-3-carboxylic acid (177 mg, 0.52 mmol) and HATU
(271 mg, 0.71 mmol) at room temperature. The mixture was stirred at
room temperature for another 2 h and quenched with the addition of
water (20 mL). The resulting mixture was extracted with ethyl acetate
(2 × 20 mL). The combined organic layers were washed with brine,
dried over sodium sulfate, and concentrated under reduced pressure
to afford crude 9H-fluoren-9-ylmethyl N-[6-[2-[2-[[5-(6-but-3-enyl-
7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-2-methoxy-pyridine-3-
carbonyl]amino]ethoxy]ethoxy]hexyl]carbamate (750 mg) as a
3
2
ylate (300 mg, 75% yield) as a yellow solid. LC-MS: m/z 531.1 (M
H) .
+
+
M
DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
brown oil. This crude was used in the next step without further
purification. LC-MS: m/z = 748.3 (M+H) .
Bergeron and Tom Pillow are acknowledged for assistance
during manuscript preparation. Grady Howes, Jan Seerveld,
Hao Zheng, Peter Sandy, Gigi Yuen, and Jeff Blaney are also
recognized for help with compound management and logistics.
Finally, we thank Catherine Wilson and the Genentech gCSI
team for profiling 27 in terms of its antiproliferative activity
against cancer-cell lines.
+
To a solution of 9H-fluoren-9-ylmethyl N-[6-[2-[2-[[5-(6-but-3-
enyl-7-oxo-1H-pyrrolo[2,3-c]pyridin-4-yl)-2-methoxy-pyridine-3-
carbonyl]amino]ethoxy]ethoxy]hexyl]carbamate (750 mg, 0.60
mmol) in DMF (10 mL) was added piperidine (1024 mg, 12.03
mmol). The mixture was stirred at room temperature for 10 h and
then separated between ethyl acetate (30 mL) and water (10 mL).
The organic layer was discarded, and the water phase was
concentrated under reduced pressure to give N-[2-[2-(6-
aminohexoxy)ethoxy]ethyl]-5-(6-but-3-enyl-7-oxo-1H-pyrrolo[2,3-c]-
pyridin-4-yl)-2-methoxy-pyridine-3-carboxamide (120 mg, 38%) as a
brown oil. The crude material was used in the next step without
further purification.
ABBREVIATIONS USED
■
BRD4(1), first bromodomain of bromodomain-containing
protein 4; BRD4(2), second bromodomain of bromodomain-
containing protein 4; BRD9, bromodomain-containing protein
9
1
; BRPF1, bromodomain- and PHD-finger-containing protein
; CBP, CREB-binding-protein bromodomain; CECR2, cat-
In the dark (because of light sensitivity), N-[2-[2-(6-
aminohexoxy)ethoxy]ethyl]-5-(6-but-3-enyl-7-oxo-1H-pyrrolo[2,3-c]-
pyridin-4-yl)-2-methoxy-pyridine-3-carboxamide (100 mg, 0.19
mmol), triethylamine (385 mg, 3.80 mmol), and 6-ROX SE (300
mg, 0.48 mmol) in DMF (1.5 mL) were added to a brown bottle. The
mixture was stirred at room temperature for 10 h and concentrated
under reduced pressure. The residue was purified by preparative
HPLC (45−67.5% ACN/0.05% ammonia in water) to give the title
eye-syndrome critical-region protein 2; TAF1(1), first
bromodomain of human transcription-initiation-factor TFIID
subunit 1; TAF1(2), second bromodomain of human tran-
scription-initiation-factor TFIID subunit 1
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AUTHOR INFORMATION
Corresponding Author
E-mail: terrydc@gene.com.
ORCID
Terry D. Crawford: 0000-0003-4663-4116
Jeremy Murray: 0000-0002-2161-9326
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
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DOI: 10.1021/acs.jmedchem.8b01225
J. Med. Chem. XXXX, XXX, XXX−XXX