Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro- 1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)

Article abstract of DOI:10.1021/jm300713s

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumori-genesis and cancer cell survival stim

Full text of DOI:10.1021/jm300713s

Article
pubs.acs.org/jmc
Discovery of 7‑Methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-
dihydro‑1H‑indol-5-yl)‑7H‑pyrrolo[2,3‑d]pyrimidin-4-amine
(GSK2606414), a Potent and Selective First-in-Class Inhibitor of
Protein Kinase R (PKR)-like Endoplasmic Reticulum Kinase (PERK)
Jeffrey M. Axten,*,† Jesus R. Medina,† Yanhong Feng,† Arthur Shu,† Stuart P. Romeril,† Seth W. Grant,†
́
William Hoi Hong Li,† Dirk A. Heerding,† Elisabeth Minthorn,† Thomas Mencken,† Charity Atkins,†
Qi Liu,† Sridhar Rabindran,† Rakesh Kumar,† Xuan Hong,∥ Aaron Goetz,‡ Thomas Stanley,§
J. David Taylor,§ Scott D. Sigethy,§ Ginger H. Tomberlin,§ Annie M. Hassell,∥ Kirsten M. Kahler,∥
Lisa M. Shewchuk,∥ and Robert T. Gampe∥
†Oncology Research, Protein Dynamics DPU, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania 19426, United
States
§
∥
‡Screening and Compound Profiling, Biological Reagents and Assay Development, and Computational and Structural Chemistry,
GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27713, United States
S
* Supporting Information
ABSTRACT: Protein kinase R (PKR)-like endoplasmic
reticulum kinase (PERK) is activated in response to a variety
of endoplasmic reticulum stresses implicated in numerous
disease states. Evidence that PERK is implicated in tumori-
genesis and cancer cell survival stimulated our search for small
molecule inhibitors. Through screening and lead optimization
using the human PERK crystal structure, we discovered
compound 38 (GSK2606414), an orally available, potent, and
selective PERK inhibitor. Compound 38 inhibits PERK
activation in cells and inhibits the growth of a human tumor xenograft in mice.
ER membrane protein containing a stress-sensing domain
facing the ER lumen, a transmembrane segment, and a cytosolic
kinase domain.16−18 Increase in unfolded proteins in the ER
causes release of ER chaperones from the stress-sensing domain
of PERK, which results in its activation via oligomerization and
autophosphorylation at multiple serine, threonine, and tyrosine
residues.19−21 Upon activation, PERK phosphorylates eukary-
otic initiation factor 2α (eIF2α) at serine 51, rendering it an
inhibitor of the ribosome translation initiation complex,
consequently reducing overall protein synthesis.22 The
reduction in translation reduces the ER burden, providing
time for the cell to process or degrade the accumulated
unfolded proteins to restore ER homeostasis. Although global
protein synthesis is decreased, there is specific increased
translation of certain mRNAs, such as ATF4, which modulate
cellular survival pathways and enhance UPR function.
Interfering with PERK function in cancer cells may limit their
ability to thrive under hypoxia or nutrient deprived conditions
and lead to apoptosis or tumor growth inhibition. Although an
attractive potential target for cancer intervention, currently
there are no known inhibitors of PERK to probe the role of its
INTRODUCTION
■
Endoplasmic reticulum (ER) stress resulting from accumulation
of unfolded proteins is emerging as an important factor in
human diseases such as neurodegeneration, heart disease,
diabetes, and cancer.1 To cope with ER stress, cells activate an
evolutionarily conserved mechanism termed the unfolded
protein response (UPR), which coordinates an adaptive cellular
signaling cascade to alleviate the impact of the stress and
enhance cell survival.2−4 Under prolonged stress and in
environments in which the UPR cannot mitigate the enduring
strain, additional pathways may be activated to induce
apoptosis or autophagy.5 Impairment of the ER capability to
properly fold and process proteins can occur under conditions
of hypoxia and nutrient deprivation, situations that are
commonly found in tumors. Human tumors, including those
derived from cervical carcinomas, glioblastomas, lung cancers,
and breast cancers, show elevated levels of UPR-related
proteins when compared to normal tissues.6−9 This, together
with evidence signifying a role of the UPR in tumorigenesis,
survival, and proliferation of cancer cells, supports the UPR as
an attractive pathway for cancer drug discovery.2,10
Protein kinase R (PKR)-like ER kinase (PERK) is one of
three primary effectors of the UPR, which has a demonstrated
role in tumor growth and angiogenesis.11−15 PERK is a type I
Received: May 21, 2012
Published: July 24, 2012
© 2012 American Chemical Society
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a
catalytic function in tumor growth. Herein we describe our
discovery of potent and selective PERK inhibitors, which
demonstrate the ability to inhibit PERK activity in cells and
display growth inhibition of human tumor xenografts in mice.
We identified potent inhibitors of PERK by screening our
proprietary collection of kinase inhibitors for activity in a PERK
mediated eIF2α phosphorylation assay. It was anticipated that
this strategy would not only quickly deliver lead molecules for
medicinal chemistry but also provide added value from existing
kinase selectivity data that could influence hit prioritization. Of
several classes of inhibitors identified, the thienopyrimidine 1
(Figure 1) was attractive based on its favorable molecular
Scheme 1
a
(a) Boc2O, DMAP, CH3CN, 71%; (b) bis(pinacolato)diboron,
KOAc, PdCl2(dppf); (c) heteroaryl bromides 6−10, Suzuki−Miyaura
conditions; (d) HCl, ethanol; (e) arylacetic acid, EDC or HATU,
DIPEA, DMF.
a
Scheme 2
Figure 1. Structures and kinase data for hits identified in a PERK
screen.
weight and lipophilicity (MW = 396.4, ClogP = 3.66), potency
(PERK IC50 = 11 nM), and selectivity evident in a limited set of
kinase inhibition data. For example, of 14 kinases with assay
data available, only Aurora B (IC50 = 58 nM) and LCK (IC50
=
1023 nM) showed inhibition within 100-fold the concentration
needed for similar PERK inhibition. Follow-up screening of
related analogues within the corporate compound collection led
to our discovery of compound 2, which contained an indoline
as the core structure. The indoline 2 had increased potency
(PERK IC50 = 3.2 nM) and offered higher kinase selectivity
against Aurora B and LCK relative to the anilide 1 (e.g., Aurora
B IC50 of 457 nM vs 58 nM for 1). On the basis of this profile,
we initiated a lead optimization campaign based on indoline 2.
a
(a) conc NH4OH, 80−86%; (b) NaH, CH3I, DMF, 74%; (c) NBS,
CH2Cl2, 95%.
onto a suitable substituted furan (Scheme 3). Hydroxylation of
1,5-diacetylindoline 13 was performed by a halogenation,
nucleophilic displacement, and hydrolysis sequence to give 14.
Reaction of 14 with malononitrile formed the substituted furan
15, which was then converted to the furanopyrimidine 16.
CHEMISTRY
■
A convergent synthetic route was devised to allow for flexible
variation of three components of the molecules: the hinge
binder heterocyclic system, the indoline core, and the
arylacetamide. We synthesized indoline boronate derivative 4a
in two steps from commercially available 5-bromoindoline 3
(Scheme 1). This building block allowed for diverse
functionalization and rapid synthesis of analogues. Suzuki−
Miyaura coupling of 4a with heteroaryl bromides 6−10 gave
intermediate 5a. Boc group removal under acidic conditions
liberated the indoline, which was then coupled with arylacetic
acids to give the target compounds represent by 5b.
Alternatively, the boronate 4b was synthesized first and
subjected to Suzuki−Miyaura coupling to afford 5b.
a
Scheme 3
The heteroaryl bromides 6, 8, and 9 are commercially
available or have been previously described. Compounds 7 and
10 were prepared as shown in Scheme 2 from commercially
available intermediates 11 and 12.
We were unable to efficiently prepare the 3-bromofuranopyr-
imidine hinge binder for use in the Suzuki−Miyaura coupling.
Therefore, for this analogue we annulated the pyrimidine ring
a
(a) pyridine·HBr3, THF, 93%; (b) NaOAc, then NaOH, 80%; (c)
malononitrile, Et2NH, DMF, 80%; (d) (EtO)2CHOAc, 1,4-dioxane,
76%; (e) NH3, MeOH, 90%; (f) KOH, EtOH, DMSO, 47%; (g)
HATU, (2,5-difluorophenyl)acetic acid, DIPEA, DMF, 32%.
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Hydrolysis of the acetamide gave the indoline which was
coupled with (2,5-dfluorophenyl)acetic acid to provide
compound 25.
Table 1. PERK Inhibitory Data for Thieno[3,2-c]pyridin-4-
amine Analogues
Direct iodination was used as a means to introduce a variety
of substituents at the 7-postion of the bicycloheteroaryl ring
(Scheme 4). Transition metal mediated coupling and
deprotection (if necessary) afforded analogues 2 and 19−21.
a
Scheme 4
a
(a) NIS, DMF, 93%; (b) R-pinacol boronate, PdCl2(dppf)−CH2Cl2
adduct, NaHCO3, dioxane−H2O, 53−85%; (c) 3,6-dihydro-2H-
pyridine-1-N-Boc-4-boronic acid pinacol ester, PdCl2(dppf)−CH2Cl2
adduct, NaHCO3, dioxane−H2O, 85%; (d) 10% Pd/C, THF, EtOH,
40 bar of H2, 41%; (e) TFA, DCM, 47%.
RESULTS AND DISCUSSION
■
Lead Optimization and SAR. At the initiation of our
research, there was no structural data available for PERK. On
the basis of kinase inhibitor structural motifs, we presumed that
the thienopyridine of 2 acted as a kinase β-strand hinge binder
and that the indoline core served as a scaffold to orient the
arylacetamide toward a back pocket of the PERK ATP binding
site, enabling productive interactions with the protein. We
began our optimization of 2 by interrogating the importance of
the pyridine substituent, which we believed was oriented
toward solvent based on this model. A small increase in
potency was realized with 4-pyridyl substitution (18, PERK
IC50 = 1.2 nM, Table 1) or with pyrazole replacements (20 and
21, PERK IC50 of 0.9 and 1.3 nM). Interestingly, we could
substitute this position with an aliphatic cyclic amine with little
effect on PERK potency. For example, the piperidine analogue
19 maintained PERK activity (IC50 = 1.9 nM) and offered a
path to increasing compound solubility and polarity. Although
removing the pyridyl group resulted in ∼4-fold loss of potency,
we were encouraged by the increased ligand efficiency of 22
(PERK IC50 = 11.7 nM, LE = 0.39). Reintroducing the 2,5-
difluoro substitution found in the screening hit gave compound
23 which was a slightly more active PERK inhibitor than 2 with
increased ligand efficiency (LE of 0.39 vs 0.34), an attractive
attribute to consider in the optimization of oral drugs.24 In
addition, we also observed some improvement in kinase
selectivity without the aromatic group at R2 (data not
shown). This is consistent with the presumed hinge binding
motif in which the aryl group fills the lipophilic plug conserved
among most kinases.25 By elimination of this interaction, the
a
Inhibitory activity as measured by cytoplasmic PERK domain
phosphorylation of EIF2α. Average values are reported with standard
deviation (see Experimental Section for details). LE = ligand
b
efficiency, defined as ΔG/Nnon‑hydrogen atoms, where ΔG ≈ −RT ln(IC50).
c
ND = not determined (data reported from a single experiment).
specificity for PERK improved because of reliance on specific
interactions deep in the PERK binding site (see discussion
below).
The thieno[3,2-c]pyridin-4-amine analogues were quite
lipophilic and generally displayed potent P450 inhibition,
high metabolic turnover, and poor pharmacokinetic parameters
(data not shown). Therefore, we set out to increase the overall
polarity of the molecules through variation of the hinge binding
heteroaryl group (Table 2). Exchanging a nitrogen for carbon
in 23 gave the thieno[2,3-d]pyrimidin-4-amine 24, lowering the
ClogP by 0.6 units to 4.1 and improving PERK activity 4-fold.
Further reductions in ClogP were accomplished by varying the
type and placement of heteroatoms. The furo[2,3-d]pyrimidin-
4-amine 25, furo[3,2-c]pyridin-4-amine 26, and pyrazolo[3,4-
d]pyrimidin-4-amine 27 analogues were less active than the
thieno derivatives 23 and 24. However, the pyrrolo[2,3-
d]pyrimidin-4-amine 28 afforded a 6-fold increase in potency
relative to 23, at the same time lowering the ClogP by 1.2 units.
The reduction in lipohilicity of 25, 27, and 28 corresponded
with improved rat blood clearance within a desirable range
below hepatic blood flow. Since all of these hinge binder
analogues theoretically meet size and shape requirements for
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Table 2. PERK Inhibition and Pharmacokinetic Data for Hinge Binder Analogues
a
b
c
compd
X
Y
Z
PERK IC50 (nM)
ClogP
rat Clb (mL min−1 kg−1
)
d
23
24
25
26
27
28
CH
CH
CH
CH
N
S
CH
N
2.5 0.67
0.6 ND
7.4 1.12
8.1 ND
5.4 1.92
0.4 0.10
4.7
4.1
3.4
4.0
2.9
3.5
ND
S
42.7
26.3
89.7
31.0
21.2
O
O
N
CH
N
N-CH3
N-CH3
CH
N
a
Inhibitory activity as measured by cytoplasmic PERK domain phosphorylation of EIF2α. Average values are reported with standard deviation (see
b
Experimental Section for details). Calculated using BioByte cLogP (the calculated logarithm of the 1-octanol−water partition coefficient of the
nonionized molecule, see www.biobyte.com). Average in vivo blood clearance determined after iv infusion in SD rats (n = 2). Not determined.
c
d
Figure 2. Crystal structure of compound 22 bound to the PERK kinase domain: (a) close-up view of 22 (green sticks) in the PERK active site
(brown cartoon and sticks); (b) close-up view of 22 bound to the PERK active site with surface rendering. The indoline fills a narrow channel
between M887 and D954 of the inward facing DFG motif. The aryl ring occupies a portion of a large lipophilic pocket. Protein residues and
structural motifs are labeled. Atomic interactions are indicated with yellow lines and distances reported in angstrom.
PERK binding, the SAR reveals that additional forces such as
electrostatics are integral to optimal PERK inhibition.
Y653, and I885. From the structure it was apparent that unfilled
hydrophobic space was available above the aryl ring meta-
position.
During our optimization, we determined the crystal structure
of 22 bound to the human PERK kinase domain.23 The PERK
structure is consistent with the global canonical fold of kinase
domains where the N- and C-terminal lobes are connected by
the intervening hinge β-strand that forms part of the ATP or
inhibitor binding cleft (Figure 2). Adjacent to the inhibitor
binding site, the catalytic lysine, K621, forms a salt bridge with
D954 of the inward facing DFG motif (Figure 2a).
Respectively, residues Q888 and C890 of the hinge β-strand
accept and donate hydrogen bonds to the inhibitor’s thieno-
[3,2-c]pyridin-4-amine moiety. The indoline ring system fills a
narrow channel between the lipophilic gatekeeper, M887, and
the inward facing DFG motif, orienting the carboxamide for
favorable interactions. The carboxamide carbonyl participates in
a water mediated hydrogen bond to the backbone carbonyl of
V952 and the NH of V651 (Figure 2b). The acetamide aryl ring
is contained by a large lipophilic pocket formed in part by the
side chains from the M887 gatekeeper, the Cα-helix L642, and
F955 of the DFG motif (Figure 2b) as well as L645, I650,
Utilizing the PERK crystal structure and analysis of 22 bound
as a design aid, we prepared numerous substituted
arylacetamides to probe the SAR of this moiety. Table 3
provides data for selected analogues in our study. In
comparison to the 2,5-difluorophenyl analogue 28 (PERK
IC50 = 0.4 nM), the ortho- and meta-monofluorinated
compounds 29 and 30 showed little difference in potency
(PERK IC50 of 0.7 and 0.2 nM, respectively) but were 2- to 5-
fold more active than the para-fluoro analogue 31 (PERK IC50
= 1.5 nM). Likewise, the activity was maintained with ortho-
and meta-chlorine substitution in 35 and 36, with a slight
preference for the meta-chloroacetamide 36 (PERK IC50 of 0.2
nM vs 0.9 nM for 35). The ortho- and meta-CH3 analogues 32
and 33 were equipotent (PERK IC50 = 0.2 nM); however, the
para-CH3 analogue 34 (PERK IC50 = 17.4 nM) lost a
significant amount of activity, likely because of steric contacts
with the protein. These results are consistent with the structural
data, which indicates that the small, tight space between the
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Table 3. Summary of SAR for Arylacetamides of the Pyrrolo[2,3-d]pyrimidin-4-amine Series
a
b
c
compd
R1
R2
R3
R4
PERK IC50 (nM)
ClogP
rat Clb (mL min−1 kg−1
)
28
29
30
31
32
33
34
35
36
37
F
H
H
F
H
F
0.4 0.10
0.7 0.13
3.5
3.4
3.4
3.4
3.7
3.7
3.7
3.9
3.9
3.2
4.11
4.3
3.6
21.2
46.4
49.6
16.5
71.2
d
d
F
H
H
F
H
H
H
H
H
H
H
H
H
H
F
e
H
H
CH3
H
H
Cl
H
H
H
H
F
0.2 ND
H
H
1.5 1.19
e
H
H
CH3
H
H
H
H
H
H
0.2 ND
e
CH3
H
0.2 ND
110.1
e
e
17.4 ND
ND
e
H
0.9 0.28
ND
e
Cl
0.2 ND
46.7
e
OCH3
CF3
CF3
F
0.5 0.12
0.4 0.32
0.3 0.32
0.2 0.01
ND
d
38 (GSK2606414)
21.5
d
39
40
17.7
F
8.3
a
Inhibitory activity as measured by cytoplasmic PERK domain phosphorylation of EIF2α. Average values are reported with standard deviation (see
b
Experimental Section for details). Calculated using BioByte cLogP (the calculated logarithm of the 1-octanol−water partition coefficient of the
nonionized molecule, see www.biobyte.com). Unless noted, rat clearance data reported were obtained by cassette dosing (up to three compounds)
as an iv infusion (2 mg/kg target dose). Discrete dosing (n = 2) as an iv infusion (2 mg/kg target dose). ND = not determined.
c
d
e
Figure 3. Crystal structure of 38 bound to PERK kinase domain: (a) close-up view of 38 (magenta sticks) in the PERK active site (brown cartoon
and sticks); (b) close-up view of 38 superimposed upon the structure of 22 (green sticks) bound to the PERK active site with surface rendering.
PERK accommodates a meta-CF3 substituent that fills the lipophilic space above the aryl ring but does not provide room for para-substitution.
Protein residues and structural motifs are labeled. Atomic interactions are indicated with dashed yellow lines with distances reported in angstrom.
Y653 side chain and A643 of the C-α helix is not likely to
accommodate a bulky para-substituent on the arylacetamide.
Electron donating and withdrawing groups were tolerated, as
demonstrated by the activity of the meta-methoxy and meta-
trifluoromethyl compounds 37 and 38 (PERK IC50 of 0.5 and
0.4 nM, respectively). An X-ray structure of 38 bound to PERK
was solved (Figure 3) and shows similar features and
interactions as those described for the thieno[3,2-c]pyridin-4-
amine 22. Furthermore, the structure reveals that the
trifluoromethyl meta-substituent occupies the lipophilic volume
bounded by the side chains of C-α helix residues, L642, A643
and also Y653, I885, and the aryl ring lies between the side
chains of L642 and the M887 gatekeeper.
During the course of our optimization, we evaluated the in
vivo rat blood clearance of analogues to gain insight into the
influence of the arylacetamide substitution pattern on
metabolism. An increase in clearance was observed in the
potent compounds 32 and 33, consistent with additional
oxidative metabolism of the exposed methyl groups (Table 3).
A significant decrease in rat blood clearance was measured for
the para-fluorophenyl analogue 31 (Clb = 16.5 mL min−1 kg−1),
in contrast to the ortho- and meta- substituted analogues 29
and 30 which displayed moderate to high clearance (Clb of 46.4
and 49.6 mL min−1 kg−1, respectively). These data implied that
the phenyl para-position was a metabolic liability and likely a
location of aromatic oxidation. Blocking the suspected
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oxidation site was not an option because para-substitution was
detrimental to PERK inhibitory activity. Therefore, we pursued
a strategy of reducing the electron density of the aryl ring to
decrease susceptibility to aromatic oxidation. The 3-trifluor-
omethylphenylacetamide 38 and 5-fluoro-3-trifluoromethylphe-
nylacetamide 39 maintained moderate rat clearance in the
range of 17−19 mL min−1 kg−1. The 2,3,5-trifluorophenylace-
tamide 40 exhibited a dramatic decrease in clearance (Clb = 8.3
mL min−1 kg−1), confirming that we could indeed influence the
pharmacokinetic properties by arylacetamide modification
while maintaining potent PERK inhibitory activity.
Characterization of Cell Activity. Compounds in this
study with potent PERK activity were routinely screened for
the ability to affect PERK function in cells. Analogues were
evaluated for their ability to prevent PERK activation upon
treatment of A549 cells with thapsigargin, a chemical inducer of
the UPR. Activity was assessed in a gel-based assay measuring
inhibition of thapsigargin induced autophosphorylation of
PERK in the presence of 0.03, 0.1, and 0.3 μM inhibitor.
Western blots for compounds 2, 21, and 38 are shown in
Figure 4 as representative examples. Compound 38 completely
Table 4. Compounds Inhibit PERK Autophosphorylation in
A459 Cells
a
compd
phospho-PERK IC50 (μM)
2
0.1−0.3
0.03−0.1
>0.3
21
22
28
31
36
38
40
0.03−0.1
0.1−0.3
<0.03
<0.03
0.03−0.1
a
IC50 estimated from a Western blot three-point dose response. See
Experimental Section for details.
Table 5. EIF2AK Selectivity Data for PERK Inhibitors
EIF2AK3 (PERK)
EIF2AK1 (HRI)
EIF2AK2 (PKR)
a
b
b
compd
IC50 (nM)
IC50 (nM)
IC50 (nM)
38
21
36
2
0.4
1.3
420
1863
214
696
978
0.2
291
3.2
12080
17020
>10000
>10000
22
11.7
a
Inhibitory activity as measured by cytoplasmic PERK domain
phosphorylation of EIF2α. Values reported are the average of at
b
least two experiments (see Experimental Section for details). Data
from a single 10-point dose response performed by Reaction Biology
Corp. (http://www.reactionbiology.com).
to trap an inactive PERK conformation. This is supported by
(1) the recently published structure26 of active mouse PERK
revealing an active site pocket that could not accommodate our
inhibitors, although they are similarly active against the mouse
protein (data not shown), and (2) our inhibitors also exhibit
time-dependent PERK inhibition and extremely slow off-rates,
consistent with binding to an inactive kinase conformation.27
On the basis of the EIF2AK selectivity profile, cellular
potency, and rat blood clearance, compound 38 was screened
against a panel of 294 kinases to further evaluate selectivity.
Overall, good selectivity was observed, with only 20 protein
kinases inhibited >85% by 38 at 10 μM.28 We subsequently
determined IC50 values for each of those kinases (Table 6). The
most sensitive kinase (c-kit, IC50 = 154 nM) is 385-fold less
sensitive than PERK (IC50 = 0.4 nM), confirming the
outstanding selectivity of compound 38.
Figure 4. Representative Western blots for compounds 2, 21, and 38
showing a range of PERK inhibition in A549 cells. Cells were
incubated with PERK inhibitor and then stimulated with thapsigargin.
After 2 h, the cells were lysed and analyzed by Western blot for
inhibition of PERK autophosphorylation. See Experimental Section for
details.
Pharmacokinetics.29 Pharmacokinetic profiling was per-
formed in mouse, rat, and dog for compound 38 (Table 7).
Compound 38 was well absorbed providing good exposure,
high oral availability, and low to moderate blood clearance in
mouse, rat, and dog. Estimated volume of distribution at steady
state was moderate to high in the three species.
inhibits PERK phosphorylation at the lowest concentration in
this assay. Compounds 21 and 2 require higher concentrations
to fully inhibit PERK phosphorylation, and these data were
used to estimate IC50 values for PERK autophosphorylation.
Table 4 contains estimated IC50 data for selected compounds
with potent PERK activity and low to moderate rat blood
clearance. All compounds inhibit activation of PERK at levels
consistent with the in vitro biochemical data.
Kinase Selectivity Profiling. Since PERK is a member of
the eIF2α-kinase (EIF2AK) family, selected PERK inhibitors
were assayed against HRI and PKR (available kinases within the
EIF2AK family) to assess selectivity (Table 5). All compounds
tested were selective and displayed at least 100-fold selectivity
over the other EIF2AKs assayed. The high specificity for PERK
within the EIF2AK family is impressive and was not predicted
based on the high similarity of the active sites. We hypothesize
that the high selectivity arises from the ability of the inhibitors
Efficacy in Human Tumor Xenograft Experiments.29
Compound 38 was advanced to an efficacy study to gauge
effects of a PERK inhibitor on the growth of tumors in mice.
Compound 38 was administered orally twice a day for 21 days
to mice bearing subcutaneous pancreatic human BxPC3
tumors. Tumor growth was measured over the course of the
study and is presented in Figure 5. When compared to vehicle
control, compound 38 inhibited tumor growth in a dose-
dependent manner, demonstrating 20% and 59% tumor growth
inhibition at doses of 50 and 150 mg/kg b.i.d. × 21 days,
respectively. Furthermore, the compound was well tolerated
with no overt signs of toxicity or changes in body weight.
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a
Table 6. Kinase Inhibitory Data for Compound 38
kinase
IC50 (nM)
154
kinase
IC50 (nM)
c-kit
RET
LCK
1215
1250
1402
1569
1578
1756
2705
2962
3195
3892
b
Aurora B
407
BRK
412
NEK4
MLK2/MAP3K10
c-MER
452
KHS/MAP4K5
MLK1/MAP3K9
TRKA
474
DDR2
524
MLCK2/MYLK2
IKKe/IKBKE
TRKC
701
AXL
1064
1115
1140
TRKB
YES/YES1
WNK2
MLK3/MAP3K11
a
Unless noted, data obtained from a single 10-point dose response
performed by Reaction Biology Corp. (http://www.reactionbiology.
b
com). Average of at least two experiments from an internal kinase
assay.
CONCLUSIONS
■
Figure 5. Antitumor activity of 38 in a human pancreatic tumor
xenograft model. BxPC3 tumor cells were injected subcutaneously in
the flank of female nude mice. Mice with established tumors (∼200
mm3) were randomized into treatment groups (n = 8 mice/group) and
administered 38 orally at 50 and 150 mg/kg b.i.d. for 21 days. Tumor
volume measurement and statistical analysis is described in the
Experimental Section.
We have discovered a series of first-in-class PERK inhibitors.
Optimization of biochemical activity and pharmacokinetics of
the indoline series was achieved by individual improvements to
each structural component of the inhibitor. The β-strand hinge
binder, indoline ring, and arylacetamide groups of the inhibitors
each participate in favorable interactions with PERK as
determined by crystallographic studies and SAR. Throughout
our investigation, the electronic nature of the aromatic ring
systems was utilized as a means to positively influence potency
and pharmacokinetics. We established that these PERK
inhibitors are functionally active in cells and inhibit PERK
signal transduction. Moreover, compound 38 was advanced to a
human xenograft efficacy study in mice and demonstrated that
small molecule inhibition of PERK can inhibit tumor growth in
vivo, consistent with the slow growth of tumors derived from
transformed PERK−/− mouse embryonic fibroblasts.6,30 Further
optimization offering improvements to the physical properties
and pharmacokinetics of 38 will be the subject of a future
communication.
purchased from Invitrogen (catalogue no. PV5106, www.invitrogen.
com). 6-His-full-length human eIF2α was purified from baculovirus
expression in Sf9 insect cells. The eIF2α protein was buffer exchanged
by dialysis into PBS, chemically modified by NHS-LC-biotin and then
buffer exchanged by dialysis into 50 mM Tris, pH 7.2, 250 mM NaCl,
5 mM DTT. Protein was aliquoted and stored at −80 °C. Quench
solution was freshly prepared and when added to the reaction gives
final concentrations of 4 nM eIF2α phospho-ser51-antibody
(purchased from Millipore, catalogue no. 07-760, www.millipore.
com), 4 nM Eu-1024 labeled anti-rabbit IgG (purchased from Perkin-
Elmer, catalogue no. AD0083), 40 nM streptavidin Surelight APC
(purchased from Perkin-Elmer, catalogue no. AD0201), and 15 mM
EDTA. Reactions were performed in black 384-well polystyrene low
volume plates (Grenier, catalogue no. 784076) in a final volume of 10
μL. The reaction volume contains, in final concentrations, 10 mM
HEPES, 5 mM MgCl2, 5 μM ATP, 1 mM DTT, 2 mM CHAPS, 40
nM biotinylated 6-His-eIF2α, and 0.4 nM GST-PERK (no. 536-1116).
Compounds under analysis were dissolved in DMSO to 1.0 mM
and serially diluted 1−3 with DMSO through 11 dilutions. An amount
of 0.1 μL of each concentration was transferred to the corresponding
well of an assay plate. This creates a final compound concentration
range from 0.00017 to 10 μM.
Because of its potency and superb target selectivity, the
PERK inhibitor 38 (GSK2606414) will serve as an excellent
tool compound to dissect the UPR and define the role of PERK
catalytic activity in a variety of biological systems spanning
multiple therapeutic areas.1
EXPERIMENTAL SECTION
PKR-like Endoplasmic Reticulum Kinase (PERK) Assay (HTRF
Format). GST-PERK (no. 536-1116) cytoplasmic domain was
■
a
Table 7. Pharmacokinetic Parameters of Compound 38
c
d
e
mouse
rat
dog
iv dose (mg/kg)
AUC0−t (ng·h/mL)
CLb (mL min−1 kg−1
Vdss (L/kg)
1.7
2.1
2.7
839.1 (732.9−945.3)
31.8 (25.8−37.8)
5.1 (4.2−5.9)
2.5 (2.0−2.9)
18.3 (suspension)
1602.1 (1439.6−1764.5)
21.5 (19.3−23.6)
3.8 (3.4−4.2)
2.5 (2.0−2.9)
4.2
3155.3 (2653.0−4127.8)
12.5 (7.8−14.9)
8.2 (8.0−8.5)
10.6 (8.0−13.8)
4.1
)
T1/2 (h)
oral dose (mg/kg)
AUC0−t (ng·h/mL)
oral F (%)
6017.1 (4186.6−7847.6)
6575.8 (4825.9−8325.7)
∼100
3848.5 (2088.9−7094.8)
72 (52−100)
b
62
a
b
Data are reported as the mean, with ranges provided in parentheses. Bioavailability (F) was estimated using mean AUC0−t values because of the
c
d
noncrossover study design. Mouse iv: 1% DMSO and 8% Captisol in saline, pH 4. Oral suspension: 0.5% HMPC/0.1% Tween 80 (aq), pH 4. Rat
iv: 1% DMSO and 8% Captisol in saline, pH 4 (n = 2). Oral solution: 1% DMSO and 20% PEG 400 in water, pH 4 (n = 2). Dog iv: 1% DMSO/
e
20% Captisol in saline, pH 5.69 (n = 3). Dog po: 1% DMSO/40% PEG 400 in water, pH 6.11 (homogeneous suspension, n = 3).
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GST-PERK solution was added to assay plates containing
compounds and preincubated for 30 min at room temperature. The
reaction was initiated by the addition of ATP and eIF2α substrate
solution. After 1 h of incubation, the quench solution was added. The
plates were covered for 2 h at room temperature prior to
determination of signal. The resulting signal was quantified on a
Viewlux reader (PerkinElmer). The APC signal is normalized to the
europium signal by transforming the data through an APC/Eu
calculation.
The data for concentration response curves were plotted as %
inhibition calculated with the data reduction formula 100 × [1 − (U1
− C2)/(C1 − C2)] versus concentration of compound where U is the
unknown value, C1 is the average control value obtained for 1%
DMSO, and C2 is the average control value obtained for 0.1 M EDTA.
Data were fitted with a curve described by
6,31 8,31 and 932 were prepared as previously described. Air or
moisture sensitive reactions were carried out under a nitrogen or argon
atmosphere. Anhydrous solvents were obtained from Sigma-Aldrich
and used as received. Flash chromatography was performed using silica
gel under standard techniques or with silica gel cartridges on an
Analogix flash chromatography instrument. NMR spectra were
recorded on a Bruker 400 MHz spectrometer. Chemical shifts (δ)
are quoted in parts per million (ppm) relative to an internal solvent
reference. Coupling constants (J) are recorded in hertz. LC−MS
analyses were performed on a Sciex or Agilent open access instrument.
Analytical HPLC data were recorded with an analytical C18 column,
eluting with an acetonitrile/water (+0.1% TFA) gradient over 4 min.
Compounds were detected by UV (254, 214, and 333 nm). All final
compounds with reported biological data were determined to be >95%
pure based on LC−MS, NMR, and analytical HPLC data unless
otherwise noted.
Y = A + [(B − A)/(1 + (10x/10c)D)]
1,1-Dimethylethyl 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaboro-
lan-2-yl)-2,3-dihydro-1H-indole-1-carboxylate (4a). To a stirred
solution of 5-bromo-2,3-dihydro-1H-indole (30 g, 151 mmol) and
DMAP (0.4 g, 3.27 mmol, 0.02 equiv) in 150 mL of CH3CN at room
temperature was added Boc2O (43 g, 197 mmol, 1.3 equiv) in one
portion. The mixture was stirred at room temperature. After 10 min,
the mixture gradually became a suspension. After 3 h, the suspension
was filtered. The cake was washed with cold CH3CN (60 mL) and
dried under house vacuum for 5 h to give 1,1-dimethylethyl 5-bromo-
2,3-dihydro-1H-indole-1-carboxylate (∼28.5 g prior to drying). The
filtrate was concentrated in vacuo, and the residue was partitioned
between CH2Cl2 (300 mL) and water (250 mL). The organic was
dried over Na2SO4, filtered, and concentrated in vacuo to give a thick
oil (∼30 g). This material was purified by silica gel chromatography
using gradient elution of 1% EtOAc in hexane to 50% EtOAc in
hexane. The combined fractions containing product were concen-
trated, and the solid residue was triturated in cold CH3CN (25 mL).
The resulting paste was filtered, and the cake was washed with cold
CH3CN (5 mL), combined with the above lot, and then dried under
vacuum at room temperature for 18 h to give 1,1-dimethylethyl 5-
bromo-2,3-dihydro-1H-indole-1-carboxylate (32.14 g, 71%). LC−MS
where A is the minimum response, B is the maximum response, D is
the slope factor, x is the concentration of the compound, and C is the
IC50. The results for each compound were recorded as IC50 values.
PERK Phosphorylation in A549 Cells. Exponentially growing
A549, a human lung adenocarcinoma cell line, was plated at 500 000
cells/well in a six-well dish in RPMI-1640 medium supplemented with
10% fetal bovine serum (FBS) and incubated overnight at 37 °C, 5%
CO2. The following morning, cells were treated with DMSO or
compound (3-fold dilutions 0.33, 0.11, 0.37 μM) and incubated as
described above for 1 h. Cells were then stimulated with 1 μM
thapsigargin for an additional hour to induce ER stress. Cells pellets
were collected and lysed in cold RIPA buffer [150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, 0.25% sodium deoxycholate, 1% NP-40, protease
and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN), and
100 mM sodium orthovanadate (Sigma Aldrich)]. Clarified lysates
were resolved by SDS−PAGE and transferred to nitrocellulose
membrane using Invitrogen’s NuPAGE system. Blots were incubated
with primary antibodies to total PERK (R&D Systems) and total eIF-
2α (Cell Signaling Technologies). IRDye700DX-labeled goat anti-
mouse IgG and IRDye800-CW donkey anti-goat IgG (LI-COR
Biosciences, Lincoln, NE) were used as secondary antibodies. Proteins
were detected on the Odyssey infrared imager (LI-COR Biosciences,
Lincoln, NE).
Pharmacokinetic Studies. All studies were conducted after
review by the Institutional Animal Care and Use Committee at GSK
and in accordance with the GSK Policy on the Care, Welfare and
Treatment of Laboratory Animals. Pharmacokinetics were studied in
male Sprague−Dawley rats, male beagle dogs, and/or male CD-1 mice
following single intravenous and/or oral administration. Absolute oral
bioavailability was estimated using a crossover study design (n of 2 or
3), unless otherwise indicated. Blood samples were assayed using
protein precipitation followed by LC/MS/MS analysis, and the
resulting concentration−time data were analyzed by noncompartmen-
tal methods (WinNonlin Professional, version 4.1).
BxPC3 Human Pancreatic Xenograft Model. All studies were
conducted after review by the Institutional Animal Care and Use
Committee at GSK and in accordance with the GSK Policy on the
Care, Welfare and Treatment of Laboratory Animals. Exponentially
growing BxPC3 tumor cells (10 × 106 cells/mouse) from cell culture
were implanted subcutaneously into the right flank of female nude
mice (Charles River). Sixteen days after implantation, mice with ∼200
mm3 tumors were randomized into various treatment groups (n = 8
mice/group). Animals were orally treated with vehicle (0.5%
hydoxypropylmethylcellulose, 0.1% Tween 80 in water, pH 4.8),
compound 38 at 50 or 150 mg/kg, b.i.d. for 21 days. Tumor volume
was measured twice weekly with calipers and calculated using the
following equation: tumor volume (mm3) = (length × width)2/2.
Results are represented as percent inhibition on completion of dosing,
which is 100[1 − (average growth of drug-treated population)/
(average growth of vehicle-treated control population)]. Statistical
analysis was performed using a two-tailed t test.
t
1
(ES) m/z = 244, 242 as prominent fragments [M − Bu]+. H NMR
(400 MHz, DMSO-d6) 1.50 (s, 9 H), 3.06 (t, J = 8.7 Hz, 2 H), 3.91 (t,
J = 8.7 Hz, 2 H), 7.31 (dd, J = 8.5, 1.9 Hz, 1 H), 7.38 (s, 1 H), 7.51−
7.71 (br s, 1 H).
A mixture of 1,1-dimethylethyl 5-bromo-2,3-dihydro-1H-indole-1-
carboxylate (32 g, 107 mmol, 1 equiv), bis(pinacolato)diboron (32.7 g,
129 mmol, 1.2 equiv), PdCl2(dppf)CH2Cl2 adduct (4.38 g, 15.37
mmol, 0.05 equiv), and potassium acetate (26.3 g, 268 mmol, 2.5
equiv) in 350 mL of dioxane in a 1 L flask was evacuated and back-
flushed with nitrogen, which was repeated 5 times. The mixture was
heated at 100 °C for 18 h. The mixture was filtered through Celite,
washing with EtOAc (500 mL). The filtrate was concentrated in vacuo.
The residue was partitioned between EtOAc (700 mL) and brine (300
mL). The organic was extracted with EtOAc (200 mL). The combined
organic was dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was dissolved in CH2Cl2 and split into seven equal
portions. Each portion was absorbed onto a dryload cartridge
immediately prior to chromatography. Purification was done on 120
g silica gel cartridges using gradient elution of 1% EtOAc in hexane to
40% EtOAc in hexane. The combined product fractions were
concentrated in vacuo to give a waxy cake, which was broken up
and dried under vacuum at room temperature for 20 h to give the
product 1,1-dimethylethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-2,3-dihydro-1H-indole-1-carboxylate (4a) (30.54 g, 82%) as a light
yellow waxy solid. LC−MS (ES) m/z = 346 [M + H]+, prominent
fragment at 290 [M − tBu]+. 1H NMR (400 MHz, DMSO-d6) 1.27 (s,
12 H), 1.50 (s, 9 H), 3.05 (t, J = 8.6 Hz, 2 H), 3.91 (t, J = 8.7 Hz, 2
H), 7.43−7.52 (m, 2 H), 7.58−7.80 (br s, 1 H).
5-Bromothieno[2,3-d]pyrimidin-4-amine (7). A suspension of
5-bromo-4-chlorothieno[2,3-d]pyrimidine (1 g, 4.01 mmol) in
concentrated aqueous ammonium hydroxide (150 mL) was stirred
overnight at 90 °C in a sealed steel vessel. The mixture was allowed to
cool to room temperature and filtered. The white solid in the filter was
Chemistry. Unless otherwise noted, commercially available
materials were used without further purification. Heteroaryl bromides
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1
air-dried to afford 5-bromothieno[2,3-d]pyrimidin-4-amine (796 mg,
86%). H NMR (400 MHz, DMSO-d6) 8.32 (s, 1 H), 7.78 (s, 1 H),
pyridin-4-amine (22) as an off-white solid (280 mg, 35%). H NMR
(400 MHz, DMSO-d6) 8.16 (d, J = 8.3 Hz, 1 H), 7.82 (d, J = 5.6 Hz, 1
H), 7.41 (s, 1 H), 7.19−7.38 (m, 8 H), 5.41 (br s, 2 H), 4.25 (t, J = 8.6
Hz, 2 H), 3.89 (s, 2 H), 3.23 (t, 2 H). LC−MS (ES) m/z = 386.0 [M
+ H]+.
1
6.38−8.22 (broad s, 2H). LC−MS (ES) m/z = 229.9, 231.9 [M + H]+.
5-Bromo-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine
(10). To 4-chloro-1H-pyrrolo[2,3-d]pyrimidine (15.2 g, 99 mmol) in
DMF (100 mL) at 0 °C was added 60% NaH (5.15 g, 129 mmol)
portionwise. After H2 bubbling stopped, iodomethane (6.81 mL, 109
mmol) was added dropwise, and then the reaction mixture was
allowed to warm to room temperature. After 3 h, the reaction mixture
was poured slowly onto water (∼800 mL) (Caution: H2 evolution due
to quenching excess NaH). The resulting solid was filtered, washed
with water followed by hexanes, and dried to afford 4-chloro-7-methyl-
3-[1-(Phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(3-
pyridinyl)thieno[3,2-c]pyridin-4-amine (2). To a solution of 3-[1-
(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]pyridin-4-
amine (22) (150 mg, 0.389 mmol) in DMF (3.0 mL) cooled in an ice-
bath was added NIS (96 mg, 0.428 mmol). The reaction mixture was
stirred at room temperature overnight. Water was poured into the
mixture. The formed brown solid was filtered and dried to give 185 mg
(93%) of the product 7-iodo-3-[1-(phenylacetyl)-2,3-dihydro-1H-
indol-5-yl]thieno[3,2-c]pyridin-4-amine (17), which was used without
further purification. LC−MS (ES) m/z = 511.9 [M + H]+.
A 25 mL pressure tube was charged with 7-iodo-3-[1-(phenyl-
acetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]pyridin-4-amine (182
mg, 0.356 mmol), 3-pyridinylboronic acid (43.7 mg, 0.356 mmol),
[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride di-
chloromethane complex (14.53 mg, 0.018 mmol), and sodium
carbonate (75 mg, 0.712 mmol) followed by dioxane (5 mL) and
water (1 mL). The mixture was heated at 120 °C for 30 min in a
microwave reactor. Water (20 mL) and ethyl acetate (20 mL) were
added, and the layers were separated. The organic layer was washed
with brine, concentrated and the residue purified by silica gel
chromatography (0−100% EtOAc in hexanes) to afford 3-[1-
(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(3-pyridinyl)thieno[3,2-
1
7H-pyrrolo[2,3-d]pyrimidine (12.2 g, 74%) as an off-white solid. H
NMR (400 MHz, DMSO-d6) 8.64 (s, 1 H), 7.73 (d, J = 3.5 Hz, 1 H),
6.63 (d, J = 3.5 Hz, 1 H), 3.85 (s, 3 H). LC−MS (ES) m/z = 168.0,
170.0 [M + H]+.
To 4-chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (12.15 g, 72.5
mmol) in CH2Cl2 (200 mL) was added NBS (13.55 g, 76 mmol)
portionwise, and the reaction mixture was stirred overnight at room
temperature. The solvent was evaporated, and the solid was washed
with water and dried to afford 5-bromo-4-chloro-7-methyl-7H-
pyrrolo[2,3-d]pyrimidine (17 g, 95%) as an off-white solid. 1H
NMR (400 MHz, DMSO-d6) 8.67 (s, 1 H), 7.99 (s, 1 H), 3.83 (s, 3
H). LC/MS (ES) m/z = 245.9, 247.9, 248.9, 249.9 [M + H]+.
A suspension of 5-bromo-4-chloro-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidine (17 g, 69.0 mmol) in concentrated aqueous ammonium
hydroxide (150 mL) was stirred for 2 days at 100 °C in a sealed vessel.
The mixture was allowed to cool to room temperature and filtered.
The collected solid was washed with Et2O and dried to afford 5-
bromo-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (12.5 g, 80%) as
a white solid. 1H NMR (400 MHz, DMSO-d6) 8.11 (s, 1 H), 7.40 (s, 1
H), 6.73 (br s, 2 H), 3.67 (s, 3 H). LC−MS (ES) m/z = 228.9,229.9
[M + H]+.
1
c]pyridin-4-amine (2) (85 mg, 51%) as a gray solid. H NMR (400
MHz, DMSO-d6) 8.88 (d, J = 1.8 Hz, 1 H), 8.62 (dd, J = 4.8, 1.5 Hz, 1
H), 8.18 (d, J = 8.1 Hz, 1 H), 8.10 (dt, J = 8.1, 1.9 Hz, 1 H), 7.96 (s, 1
H), 7.56 (dd, J = 8.1, 4.8 Hz, 1 H), 7.50 (s, 1 H), 7.23−7.40 (m, 7 H),
5.63 (br s, 2 H), 4.26 (t, J = 8.5 Hz, 2 H), 3.90 (s, 2 H), 3.24 (t, 2 H).
LC−MS (ES) m/z = 463.1 [M + H]+.
3-[1-(Phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]-
pyridin-4-amine (22). To a mixture of phenylacetic acid (0.687 g,
5.05 mmol) and HATU (2.112 g, 5.55 mmol) in DMF (5 mL) was
added DIPEA (0.882 mL, 5.05 mmol), and the resulting mixture was
stirred for 15 min at room temperature. 5-Bromo-2,3-dihydro-1H-
indole (1 g, 5.05 mmol) was added, and the reaction mixture was
stirred at room temperature overnight. The mixture was poured onto
water, and the resulting precipitate was filtered and air-dried to afford
the 5-bromo-1-(phenylacetyl)-2,3-dihydro-1H-indole (1.24 g, 78%) as
3-[1-(Phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(4-
pyridinyl)thieno[3,2-c]pyridin-4-amine (18). A mixture of 7-iodo-
3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]pyridin-4-
amine (101 mg, 0.198 mmol), pyridine-4-boronic acid pinacol ester
(53 mg, 0.258 mmol), and PdCl2(dppf)−CH2Cl2 adduct (8 mg, 9.80
μmol) in 1,4-dioxane (1.5 mL) and saturated aqueous sodium
bicarbonate (0.6 mL, 0.600 mmol) was degassed with nitrogen for 10
min in a microwave vial. The vial was then capped, and the mixture
was stirred at 120 °C in the microwave for 30 min. The mixture was
cooled, poured into water (15 mL), and extracted with ethyl acetate (2
× 15 mL). The combined extracts were washed with brine (1 × 15
mL), dried (Na2SO4), filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (Analogix, 24 g SiO2 cartidge),
eluting with 25−100% EtOAc in hexanes gradient over 30 min, then
EtOAc for 10 min, then 0−10% MeOH in EtOAc over 20 min to give
3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(4-pyridinyl)thieno-
[3,2-c]pyridin-4-amine (18) (66 mg, 69% yield) as a beige solid. LC/
MS (ES) m/z = 463 [M + H]+. 1H NMR (400 MHz, DMSO-d6) 3.24
(t, J = 8.46 Hz, 2 H), 3.90 (s, 2 H), 4.26 (t, J = 8.46 Hz, 2 H), 5.74 (br
s, 2 H), 7.23−7.39 (m, 7 H), 7.52 (s, 1 H), 7.70−7.75 (m, 2 H), 8.09
(s, 1 H), 8.18 (d, J = 8.34 Hz, 0 H), 8.65−8.72 (m, 2 H).
3-[1-(Phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(4-
piperidinyl)thieno[3,2-c]pyridin-4-amine (19). A mixture of 7-
iodo-3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]-
pyridin-4-amine (298 mg, 0.583 mmol), 3,6-dihydro-2H-pyridine-1-N-
Boc-4-boronic acid pinacol ester (238 mg, 0.770 mmol), and
PdCl2(dppf)−CH2Cl2 adduct (24 mg, 0.029 mmol) in 1,4-dioxane
(6 mL) and saturated aqueous sodium bicarbonate (2 mL, 2.0 mmol)
was degassed with nitrogen for 10 min in a microwave vial. The vial
was then capped, and the mixture was stirred at 120 °C in a microwave
reactor for 30 min. LC−MS showed complete conversion to the
product. The mixture was cooled, poured into water (50 mL), and
extracted with ethyl acetate (2 × 50 mL). The extracts were washed
with brine (1 × 75 mL), dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was purified by flash chromatography (Analogix, 40
g SiO2, 25−100% EtOAc in hexanes gradient over 45 min, then EtOAc
for 5 min) to give 1,1-dimethylethyl 4-{4-amino-3-[1-(phenylacetyl)-
1
a tan solid. LC−MS (ES) m/z = 338.0,340.0 [M + Na]+. H NMR
(400 MHz, DMSO-d6) 7.98 (d, J = 8.6 Hz, 1 H), 7.42 (s, 1 H), 7.19−
7.37 (m, 6 H), 4.18 (t, J = 8.6 Hz, 2 H), 3.84 (s, 2 H), 3.16 (t, 2 H).
To 5-bromo-1-(phenylacetyl)-2,3-dihydro-1H-indole (0.658 g,
2.081 mmol), bispinacolatodiboron (0.634 g, 2.497 mmol), and
potassium acetate (0.613 g, 6.24 mmol) in a sealable tube was added
1,4-dioxane (15 mL), and the mixture was degassed with N2 for 10
min. PdCl2(dppf)−CH2Cl2 adduct (0.085 g, 0.104 mmol) was added,
and the reaction mixture was sealed and stirred for 48 h at 100 °C. The
mixture was cooled to room temperature and treated with 5 mL of
water, 3-bromothieno[3,2-c]pyridin-4-amine (0.524 g, 2.289 mmol),
and NaHCO3 (175 mg). The mixture was degassed with N2 for 10
min. PdCl2(dppf)−CH2Cl2 adduct (0.085 g, 0.104 mmol) was added,
and the reaction mixture was sealed and stirred overnight at 100 °C.
The mixture was poured onto water and ethyl acetate and then filtered.
The filtrate was transferred to a separatory funnel. The organic layer
was separated, and the aqueous layer was further extracted with ethyl
acetate. The combined organic layers were washed with brine, dried
(MgSO4), filtered, and concentrated. Flash chromatography on SiO2
(gradient 100% CHCl3 to 90:10:1 CHCl3/CH3OH/NH4OH)
afforded a few fractions containing the desired product with an
impurity. The fractions were combined and evaporated. The resulting
residue was dissolved in MeOH/CH2Cl2 (1 mL/5 mL), dry loaded
onto silica, and purified by chromatography (Analogix 25−14 g
cartridge) using a gradient of 0−100% EtOAc/hexane. The fractions
with the pure compound were combined, the solvents were
evaporated, and the resulting residue was triturated with EtOAc to
give 3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]-
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2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]pyridin-7-yl}-3,6-dihydro-
1(2H)-pyridinecarboxylate (280 mg, 85%) as a beige solid. LC/MS
(ES) m/z = 567 [M + H]+.
3.24 (t, J = 8.46 Hz, 2 H), 3.89 (s, 2 H), 3.93 (s, 3 H), 4.26 (t, J = 8.46
Hz, 2 H), 5.41 (br s, 2 H), 7.22−7.30 (m, 2 H), 7.30−7.39 (m, 5 H),
7.49 (s, 1 H), 7.88 (s, 1 H), 8.03 (s, 1 H), 8.14 (s, 1 H), 8.17 (d, J =
8.08 Hz, 1 H).
A solution of 1,1-dimethylethyl 4-{4-amino-3-[1-(phenylacetyl)-2,3-
dihydro-1H-indol-5-yl]thieno[3,2-c]pyridin-7-yl}-3,6-dihydro-1(2H)-
pyridinecarboxylate (220 mg, 0.388 mmol), ethanol (10 mL), and
THF (15 mL) was subjected to 10% Pd/C hydrogenation on an H-
Cube reactor at 40 °C and 40 bar for 23 h. After concentration in
vacuo, and the residue was dry loaded onto silica gel (1 g) and purified
by flash chromatography (Analogix, 40 g SiO2, CH2Cl2 to 95/5/0.5
CH2Cl2/MeOH/NH4OH gradient over 42 min) to give 1,1-
dimethylethyl 4-{4-amino-3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-
5-yl]thieno[3,2-c]pyridin-7-yl}-1-piperidinecarboxylate (91 mg, 41%)
as an off-white solid. LC/MS (ES) m/z = 569.2 [M + H]+.
3-[1-(Phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]-
pyridin-4-amine (22). A sealable tube was charged with 5-bromo-1-
(phenylacetyl)-2,3-dihydro-1H-indole (0.658 g, 2.081 mmol), bispina-
colatodiboron (0.634 g, 2.5 mmol), and potassium acetate (0.613 g,
6.24 mmol). 1,4-Dioxane (15 mL) was added, and the mixture was
degassed with N2 for 10 min. PdCl2(dppf)−CH2Cl2 adduct (0.085 g,
0.104 mmol) was added, and the reaction mixture was stirred for 48 h
at 100 °C. The mixture was cooled to room temperature and treated
with 5 mL of water, 3-bromothieno[3,2-c]pyridin-4-amine (0.524 g,
2.289 mmol), and NaHCO3 (175 mg). The mixture was degassed with
N2 for 10 min. PdCl2(dppf)−CH2Cl2 adduct (0.085 g, 0.104 mmol)
was added, and the reaction mixture was stirred overnight at 100 °C.
The mixture was poured onto water and ethyl acetate and was filtered.
The filtrate was transferred to a separatory funnel. The organic layer
was separated, and the aqueous layer was further extracted with ethyl
acetate. The combined organic layers were washed with brine, dried
(MgSO4), filtered, and concentrated. Flash chromatography on SiO2
(gradient 100% CHCl3 to 90:10:1 CHCl3/CH3OH/NH4OH)
afforded a few fractions containing the desired product with an
impurity. The fractions were combined and evaporated. The resulting
residue was dissolved in MeOH/CH2Cl2 (1 mL/5 mL), then dry
loaded and purified by silica gel chromatography (Analogix 25−14 g
cartridge), using a gradient of 0−100% EtOAc/hexane. The fractions
with the pure compound were combined and evaporated. The
resulting residue was triturated with EtOAc to give 3-[1-(phenyl-
acetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]pyridin-4-amine (22)
as an off-white solid (280 mg, 35%). LC−MS (ES) m/z = 386.0 [M
+ H]+. 1H NMR (400 MHz, DMSO-d6) 8.16 (d, J = 8.3 Hz, 1 H), 7.82
(d, J = 5.6 Hz, 1 H), 7.41 (s, 1 H), 7.19−7.38 (m, 8 H), 5.41 (br s, 2
H), 4.25 (t, J = 8.6 Hz, 2 H), 3.89 (s, 2 H), 3.23 (t, 2 H).
3-{1-[(2,5-Difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-
thieno[3,2-c]pyridin-4-amine (23). To a mixture of (2,5-
difluorophenyl)acetic acid (0.869 g, 5.05 mmol) and HATU (2.11 g,
5.55 mmol) in DMF (10 mL) was added DIPEA (0.882 mL, 5.05
mmol), and the resulting mixture was stirred for 15 min at room
temperature. 5-Bromo-2,3-dihydro-1H-indole (1 g, 5.05 mmol) was
added, and the reaction mixture was stirred at room temperature for 1
h. The mixture was poured onto water, and the resulting aqueous
mixture was filtered to afford 5-bromo-1-[(2,5-difluorophenyl)acetyl]-
2,3-dihydro-1H-indole (1.6 g) as a tan solid. LC−MS (ES) m/z = 376,
378 [M + Na]+. 1H NMR (400 MHz, DMSO-d6) 7.93 (d, J = 8.6 Hz, 1
H), 7.45 (s, 1 H), 7.32 (dd, J = 8.6, 1.8 Hz, 1 H), 7.12−7.29 (m, 3 H),
4.24 (t, J = 8.5 Hz, 2 H), 3.91 (s, 2 H), 3.21 (t, 2 H).
In a sealable tube, to 5-bromo-1-[(2,5-difluorophenyl)acetyl]-2,3-
dihydro-1H-indole (0.700 g, 1.99 mmol), bispinacolatodiboron (0.606
g, 2.39 mmol), and potassium acetate (0.585 g, 5.96 mmol) was added
1,4-dioxane (15 mL), and the mixture was degassed with N2 for 10
min. PdCl2(dppf)−CH2Cl2 adduct (0.081 g, 0.99 mmol) was added,
and the reaction mixture was sealed and stirred for 48 h at 100 °C. The
mixture was cooled to room temperature and treated with 5 mL of
water, 3-bromothieno[3,2-c]pyridin-4-amine (0.501 g, 2.186 mmol),
and sodium bicarbonate (167 mg, 1.988 mmol). The mixture was
degassed with N2 for 10 min. PdCl2(dppf)−CH2Cl2 adduct (0.085 g,
0.104 mmol) was added, and the reaction mixture was stirred
overnight at 100 °C. The mixture was poured onto water and ethyl
acetate, then filtered. The filtrate was transferred to a separatory
funnel, and the organic layer was separated. The aqueous layer was
further extracted with ethyl acetate, and the combined organic layers
were washed with brine, dried (MgSO4), filtered, and concentrated.
Purification by chromatography (Analogix silica gel cartridge), eluting
with a gradient of 0−100% EtOAc/hexane, gave 526 mg (62%) of 3-
{1-[(2,5-difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}thieno[3,2-
c]pyridin-4-amine (23) as an off-white solid. LC−MS (ES) m/z =
422.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) 8.11 (d, J = 8.3 Hz,
1 H), 7.82 (d, J = 5.6 Hz, 1 H), 7.42 (s, 1 H), 7.35 (s, 1 H), 7.12−7.31
TFA (0.5 mL, 6.49 mmol) was added to a suspension of 1,1-
dimethylethyl 4-{4-amino-3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-
5-yl]thieno[3,2-c]pyridin-7-yl}-1-piperidinecarboxylate (90 mg, 0.158
mmol) in CH2Cl2 (3.5 mL), and the mixture was stirred at room
temperature under nitrogen for 30 min. The reaction mixture was then
concentrated in vacuo. The residue was taken up in CH2Cl2 and
passed through a PL-HCO3 MP-resin cartridge, rinsing with more
CH2Cl2. The filtrate was concentrated in vacuo, and the residue was
purified by flash chromatography (Analogix, 24 g SiO2, CH2Cl2 to 80/
20/2 CH2Cl2/MeOH/NH4OH gradient over 30 min) to give 3-[1-
(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(4-piperidinyl)thieno-
[3,2-c]pyridin-4-amine (19) (37 mg, 47%) as a white solid. LC−MS
1
(ES) m/z = 469 [M + H]+. H NMR (400 MHz, DMSO-d6) 1.64−
1.77 (m, 2 H), 1.77−1.86 (m, 2 H), 2.57−2.77 (m, 3 H), 3.07 (d, J =
12.13 Hz, 2 H), 3.22 (t, J = 8.34 Hz, 2 H), 3.89 (s, 2 H), 4.24 (t, J =
8.59 Hz, 2 H), 5.24 (br s, 2 H), 7.19−7.39 (m, 7 H), 7.41 (s, 1 H),
7.70 (s, 1 H), 8.15 (d, J = 8.34 Hz, 1 H).
3-[1-(Phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(1H-pyra-
zol-4-yl)thieno[3,2-c]pyridin-4-amine (20). A mixture of 7-iodo-3-
[1-(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-c]pyridin-4-
amine (101 mg, 0.198 mmol), 1-Boc-pyrazol-4-boronic acid pinacol
ester (88 mg, 0.299 mmol), and PdCl2(dppf)−CH2Cl2 adduct (9 mg,
0.011 mmol) in 1,4-dioxane (2 mL) and saturated aqueous sodium
bicarbonate (0.6 mL, 0.600 mmol) was degassed with nitrogen for 10
min in a microwave vial. The vial was then capped, and the mixture
was stirred at 120 °C in a microwave reactor for 30 min. The mixture
was cooled, poured into water (15 mL), and extracted with ethyl
acetate (2 × 15 mL). The combined extracts were washed with brine
(1 × 15 mL), dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by flash chromatography (Analogix, 24 g SiO2,
50−100% EtOAc in hexanes gradient over 15 min, then EtOAc for 5
min, then 0−10% MeOH in EtOAc over 20 min) to give 3-[1-
(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-7-(1H-pyrazol-4-yl)thieno-
[3,2-c]pyridin-4-amine (20) (50 mg, 53%) as a light gray solid. LC−
MS (ES) m/z = 452 [M + H]+. 1H NMR (400 MHz, DMSO-d6) 3.24
(t, J = 8.34 Hz, 2 H), 3.89 (s, 2 H), 4.26 (t, J = 8.46 Hz, 2 H), 5.40 (br
s, 2 H), 7.22−7.30 (m, 2 H), 7.30−7.40 (m, 5 H), 7.48 (s, 1 H), 7.95
(br s, 1 H), 8.06 (s, 1 H), 8.12−8.21 (m, 2 H), 13.10 (br s, 1 H).
7-(1-Methyl-1H-pyrazol-4-yl)-3-[1-(phenylacetyl)-2,3-dihy-
dro-1H-indol-5-yl]thieno[3,2-c]pyridin-4-amine (21). A mixture
of 7-iodo-3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]thieno[3,2-
c]pyridin-4-amine (102 mg, 0.199 mmol), 1-methylpyrazole-4-boronic
acid pinacol ester (60 mg, 0.288 mmol), and PdCl2(dppf)−CH2Cl2
adduct (8 mg, 9.80 μmol) in 1,4-dioxane (2.0 mL) and saturated
aqueous sodium bicarbonate (0.6 mL, 0.600 mmol) was degassed with
nitrogen for 10 min in a microwave vial. The vial was then capped, and
the mixture was stirred at 120 °C in a microwave reactor for 30 min.
The mixture was cooled, poured into water (15 mL), and extracted
with ethyl acetate (2 × 15 mL). The extracts were washed with brine
(1 × 15 mL), dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by flash chromatography (Analogix, 24 g SiO2,
50−100% EtOAc in hexanes gradient over 10 min, then EtOAc for 5
min, then 0−10% MeOH in EtOAc over 20 min) to give 7-(1-methyl-
1H-pyrazol-4-yl)-3-[1-(phenylacetyl)-2,3-dihydro-1H-indol-5-yl]-
thieno[3,2-c]pyridin-4-amine (21) (69 mg, 71%) as a light gray solid.
LC−MS (ES) m/z = 466 [M + H]+. 1H NMR (400 MHz, DMSO-d6)
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(m, 5 H), 5.41 (br s, 2 H), 4.31 (t, J = 8.3 Hz, 2 H), 3.96 (s, 2 H),
3.23−3.31 (m, 2 H).
water and dried under house vacuum overnight to give 4-(1-acetyl-2,3-
dihydro-1H-indol-5-yl)-2-amino-3-furancarbonitrile (15) (2.22 g,
1
5-{1-[(2,5-Difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-
thieno[2,3-d]pyrimidin-4-amine (24). To a mixture of 5-bromo-1-
[(2,5-difluorophenyl)acetyl]-2,3-dihydro-1H-indole (150 mg, 0.426
mmol), bis(pinacolato)diboron (114 mg, 0.447 mmol), and potassium
acetate (125 mg, 1.278 mmol) was added 1,4-dioxane (6 mL), and the
mixture was degassed with N2 for 10 min. PdCl2(dppf)−CH2Cl2
adduct (17.39 mg, 0.021 mmol) was added, and the reaction mixture
was stirred for 3 h at 100 °C in a sealed vessel. The mixture was cooled
to room temperature. 5-Bromothieno[2,3-d]pyrimidin-4-amine (7)
(103 mg, 0.447 mmol) and saturated aqueous NaHCO3 (2 mL) were
added, and N2 gas was bubbled through the mixture for 10 min.
PdCl2(dppf)−CH2Cl2 adduct (17.39 mg, 0.021 mmol) was added.
The vessel was sealed, and the reaction mixture was stirred overnight
at 100 °C. The mixture was poured onto water, and a precipitate was
formed. The mixture was filtered, and the solid was taken up into a
mixture of 20% CH3OH/CH2Cl2. The resulting mixture was filtered,
injected into a 90 g silica gel column, and purified via flash
chromatography (gradient 100% hexanes to 100% EtOAc). The
fractions containing the product were combined and concentrated to
afford a solid. Trituration with Et2O afforded 5-{1-[(2,5-
difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}thieno[2,3-d]-
80%) as beige solids. LC−MS (ES) m/z = 268 [M + H]+. H NMR
(400 MHz, methanol-d4) 8.11 (d, J = 8.3 Hz, 1 H), 7.46 (s, 1 H), 7.40
(d, J = 8.3 Hz, 1 H), 7.10 (s, 1 H), 4.18 (t, J = 8.5 Hz, 2 H), 3.25 (t, J =
8.5 Hz, 2 H), 2.26 (s, 3 H).
To a suspension of 4-(1-acetyl-2,3-dihydro-1H-indol-5-yl)-2-amino-
3-furancarbonitrile (1.248 g, 4.67 mmol) in 1,4-dioxane (12 mL) was
added bis(ethyloxy)methyl acetate (2 mL, 12.29 mmol, 2.63 equiv) in
one portion. The resulting suspension was heated in an oil bath at 60
°C. After 15 min of heating, the mixture became a solution. Heating
was continued for 4 h, and the mixture was cooled to room
temperature. After 10 h of aging at room temperature, the mixture
became a suspension, which was filtered. The cake was washed with
hexane and dried under vacuum to give ethyl [4-(1-acetyl-2,3-dihydro-
1H-indol-5-yl)-3-cyano-2-furanyl]imidoformate (1.20 g, 76%) as tan
solids. 1H NMR (400 MHz, DMSO-d6) 1.34 (t, J = 7.1 Hz, 3 H), 2.18
(s, 3 H), 3.19 (t, J = 8.6 Hz, 2 H), 4.14 (t, J = 8.6 Hz, 2 H), 4.38 (q, J =
6.6 Hz, 2 H), 7.38−7.47 (m, 1 H), 7.49 (s, 1 H), 7.94 (s, 1 H), 8.09 (d,
J = 8.3 Hz, 1 H), 8.64 (s, 1 H).
To a homogeneous dark brown solution of ethyl [4-(1-acetyl-2,3-
dihydro-1H-indol-5-yl)-3-cyano-2-furanyl]imidoformate (2.34 g, 7.24
mmol) in 20 mL of CH2Cl2 was added 6 mL of 7 N NH3 in MeOH in
one portion. The resulting mixture was stirred at room temperature.
After 10 min, the mixture became a suspension. After 18 h, LC−MS
showed conversion complete. The suspension was concentrated in
vacuo, and the residue was dried under vacuum to give 5-(1-acetyl-2,3-
dihydro-1H-indol-5-yl)furo[2,3-d]pyrimidin-4-amine (16) (1.92 g,
90% yield) as a beige solid. LC−MS (ES) m/z = 294.9 [M + H]+.
1H NMR (400 MHz, DMSO-d6) 8.20 (br s, 2 H), 8.06 (d, J = 8.3 Hz,
1 H), 7.87 (br s, 1 H), 7.64 (s, 1 H), 7.25−7.55 (m, 2 H), 4.12 (t, J =
8.5 Hz, 2 H), 3.11−3.26 (m, 2 H), 2.17 (s, 3 H); NH2 protons not
visible.
1
pyrimidin-4-amine (120 mg, 67%) as a white solid. H NMR (400
MHz, DMSO-d6) 3.27 (t, 2 H), 3.96 (s, 2 H), 4.31 (t, J = 8.46 Hz, 2
H), 7.13−7.32 (m, 4 H), 7.37 (s, 1 H), 7.43 (s, 1 H), 8.11 (d, J = 8.08
Hz, 1 H), 8.34 (s, 1 H). LC−MS (ES) m/z = 423.1 [M + H]+.
5-{1-[(2,5-Difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-
furo[2,3-d]pyrimidin-4-amine (25). To a suspension of 1,5-
diacetylindoline (10.0 g, 49.2 mmol) in 90 mL of THF at room
temperature was added pyridinium tribromide (16.52 g, 51.7 mmol, 1
equiv) as a solid portionwise over a period of 10 min. When there was
about 1.5 g of pyridinium tribromide left, the mixture solidified.
Another 30 mL of THF was added so that the mixture could be
stirred. The remaining 1.5 g of tribromide was added in one portion,
and the mixture was stirred at room temperature. After 1.5 h, LC−MS
showed complete conversion. The suspension was filtered, and the
cake was washed with THF (2 × 30 mL) and then water (2 × 50 mL).
The wet cake was dried under house vacuum at room temperature for
2 days to give 1-(1-acetyl-2,3-dihydro-1H-indol-5-yl)-2-bromoetha-
none (12.89 g, 93%) as light gray solids. LC−MS (ES) m/z = 281.9,
283.9. 1H NMR (400 MHz, DMSO-d6, ∗ denotes minor rotamer
signal) 2.14−2.27 (m, 3 H), 3.20 (t, J = 8.5 Hz, 2 H), 4.17 (t, J = 8.5
Hz, 2 H), 4.84, 5.86* (s, 2H), 7.78−7.94 (m, 2 H), 8.05−8.18 (m,
1H).
A dark brownish suspension of 5-(1-acetyl-2,3-dihydro-1H-indol-5-
yl)furo[2,3-d]pyrimidin-4-amine (1.71 g, 5.81 mmol) and LiOH·H2O
(5.50 g, 131 mmol, 22.6 equiv) in 50 mL of EtOH and 10 mL of water
and 10 mL of DMSO was degassed and back-flushed with nitrogen.
This cycle was repeated 4×, and the mixture was heated in an oil bath
at 100 °C for 48 h. LC−MS showed there was still 22% starting
material left. To the mixture was added KOH (3.26 g, 58.1 mmol, 10
equiv) as pellets. The suspension was degassed and heated at 100 °C
for another 16 h. The mixture was cooled and filtered. The cake was
rinsed with 30 mL of EtOH, and the filtrate was cooled in an ice bath.
The pH was adjusted by adding cold 6 N HCl to pH 7−8. The
resulting brownish mixture was concentrated in vacuo to remove as
much solvent as possible. The solid residue was taken up in water to
give a suspension, which was chilled in a refrigerator, followed by
filtration. The cake was washed with water (2 × 8 mL) and dried
under house vacuum for 5 h and then under vacuum over P2O5 for 15
h to afford 5-(2,3-dihydro-1H-indol-5-yl)furo[2,3-d]pyrimidin-4-amine
(0.76 g, 47%) as dark tan-colored solids. LC−MS (ES) m/z = 252.9
A pasty suspension of 1-(1-acetyl-2,3-dihydro-1H-indol-5-yl)-2-
bromoethanone (39.0 g, 138 mmol) and sodium acetate (56.7 g,
691 mmol, 5 equiv) in EtOH (300 mL) and water (300 mL) was
heated in an oil bath at 70 °C for 3 h. The mixture was cooled in an ice
bath and then stored in the refridgerator overnight. To the cold
suspension was added cold 6 N NaOH (25 mL, 150 mmol, 1.09
equiv). The mixture was stirred in an ice bath for 2.5 h, and the cold
mixture was acidified with 2 N HCl to pH 6. The mixture was
concentrated in vacuo to about 100 mL of volume as an aqueous
slurry, which was diluted with 200 mL of water and ether (300 mL).
The suspension was stirred at room temperature for 15 min, then
filtered. The light brownish cake was washed with water (2 × 100 mL)
and dried under house vacuum overnight to give 1-(1-acetyl-2,3-
dihydro-1H-indol-5-yl)-2-hydroxyethanone (14) (24.22 g, 80%). LC−
1
[M + H]+. H NMR (400 MHz, DMSO-d6) 8.23 (s, 1 H), 7.79 (s, 1
H), 7.16 (s, 1 H), 7.05 (dd, J = 8.0, 1.6 Hz, 1 H), 6.61 (d, J = 7.8 Hz, 1
H), 5.77 (s, 1 H), 3.48 (td, J = 8.5, 1.6 Hz, 2 H), 2.98 (t, 2 H); NH2
protons not visible.
To a stirred dark brownish solution of 5-(2,3-dihydro-1H-indol-5-
yl)furo[2,3-d]pyrimidin-4-amine (360 mg, 1.43 mmol) and HATU
(597 mg, 1.57 mmol, 1.1 equiv) in 3 mL of DMF was added DIEA
(274 uL, 1.57 mmol, 1.1 equiv). To this mixture was added (2,5-
difluorophenyl)acetic acid portionwise (246 mg total, 1.43 mmol, 1
equiv) over a 1 h period. The mixture was stirred for another 2 h and
then added to 50 mL of ice−water. The resulting suspension was
filtered. The brown cake was washed with water (2 × 10 mL) and then
air-dried to give crude product (760 mg). This material was dissolved
in 10% MeOH in CH2Cl2 and adsorbed onto silica. Purification was
performed on a 60 g silica gel cartridge using gradient elution of 1% A
to 55% A in CH2Cl2 (A was a mixture of 3200 mL of CH2Cl2, 800 mL
of MeOH, and 80 mL of concentrated NH4OH). Pure fractions were
combined and concentrated in vacuo. The residue was dissolved in
10% MeOH in CHCl3 and filtered. The filtrate was concentrated in
1
MS (ES) m/z = 220.1 [M + H]+. H NMR (400 MHz, DMSO-d6)
8.10 (d, J = 8.8 Hz, 1 H), 7.66−7.88 (m, 2 H), 4.97 (t, J = 5.8 Hz, 1
H), 4.73 (d, J = 5.8 Hz, 2 H), 4.16 (t, J = 8.5 Hz, 2 H), 3.19 (t, J = 8.6
Hz, 2 H), 2.20 (s, 3 H).
To a suspension of 1-(1-acetyl-2,3-dihydro-1H-indol-5-yl)-2-
hydroxyethanone (2.29 g, 10.45 mmol) and malononitrile (759 mg,
11.49 mmol, 1.1 equiv) in DMF (16 mL) at room temperature was
added diethylamine (1.64 mL, 15.67 mmol, 1.5 equiv). The brown
suspension was stirred at ambient temperature for 2 h, during which it
became a solution and then a suspension again. The mixture was
poured into 150 mL of water, then filtered. The cake was washed with
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vacuo, and the residue was taken up in 1.5 mL of CHCl3. MTBE (1
mL) and hexane (7 mL) were added to give a suspension, which was
filtered. The cake was washed with hexane (2 × 4 mL) and then dried
under vacuum at 65 °C for 18 h to afford 5-{1-[(2,5-difluorophenyl)-
acetyl]-2,3-dihydro-1H-indol-5-yl}furo[2,3-d]pyrimidin-4-amine (25)
(295 mg, 32%) as an off-white solid. LC−MS (ES) m/z = 407 [M
+ H]+. 1H NMR (400 MHz, DMSO-d6) 3.28 (t, J = 8.6 Hz, 2 H), 3.96
(s, 3 H), 4.30 (t, J = 8.5 Hz, 2 H), 7.13−7.28 (m, 3 H), 7.30 (d, J = 9.1
Hz, 1 H), 7.40 (s, 1 H), 7.93 (s, 1 H), 8.12 (d, J = 8.3 Hz, 1 H), 8.25
(s, 1 H); NH2 protons not visible.
3-{1-[(2,5-Difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-
furo[3,2-c]pyridin-4-amine (26). A mixture of 3-bromofuro[3,2-
c]pyridin-4-amine (8) (3.0 g, 14.09 mmol), 1,1-dimethylethyl 5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-indole-1-
carboxylate (4a) (5.35 g, 15.48 mmol), and PdCl2(dppf)−CH2Cl2
adduct (0.573 g, 0.7 mmol) in 1,4-dioxane (120 mL) and saturated
aqueous sodium bicarbonate (43 mL, 43.0 mmol) was degassed with
nitrogen for 20 min. The mixture was then stirred at reflux under
nitrogen for 16 h. It was then cooled, poured into half-saturated
aqueous NaHCO3 (250 mL), and extracted with ethyl acetate (2 ×
250 mL). The combined extracts were washed with brine (1 × 250
mL), dried (Na2SO4), filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (Analogix, 400 g SiO2, 20−100%
EtOAc in hexanes gradient over 60 min, then 100% EtOAc for 15
min) to give 1,1-dimethylethyl 5-(4-aminofuro[3,2-c]pyridin-3-yl)-2,3-
dihydro-1H-indole-1-carboxylate (3.93 g, 79%) as an off-white solid.
LC−MS (ES) m/z = 352 [M + H]+. 1H NMR (400 MHz, DMSO-d6)
1.53 (s, 9 H), 3.14 (t, J = 8.59 Hz, 2 H), 3.97 (t, J = 8.72 Hz, 2 H),
5.52 (s, 2 H), 6.93 (d, J = 5.81 Hz, 1 H), 7.30 (d, J = 8.08 Hz, 1 H),
7.34 (s, 1 H), 7.83 (br s, 1 H), 7.86 (d, J = 5.81 Hz, 1 H), 7.89 (s, 1
H).
allowed to cool to room temperature and poured onto water (∼150
mL). The resulting mixture was filtered, and the collected solid was
triturated with Et2O and then taken up in a 90:10 mixture of CHCl3/
CH3OH (∼7 mL), which was then filtered. The filtrate was injected
into a 90 g SiO2 column. Flash chromatography on SiO2 (gradient
100% CHCl3 to 90:10:1 CHCl3/CH3OH/NH4OH) provided 3-{1-
[(2,5-difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-1-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (27) (160 mg, 84%) as a brown
solid. 1H NMR (400 MHz, DMSO-d6) 3.29 (t, J = 8.34 Hz, 2 H), 3.94
(s, 3 H), 3.97 (s, 2 H), 4.30 (t, J = 8.46 Hz, 2 H), 7.14−7.31 (m, 3 H),
7.44 (d, J = 8.34 Hz, 1 H), 7.53 (s, 1 H), 8.14 (d, J = 8.34 Hz, 1 H),
8.25 (s, 1 H). LC−MS (ES) m/z = 421.1 [M + H]+.
5-{1-[(2,5-Difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-
7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (28). A mixture of
5-bromo-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (10) (50.0 g,
220 mmol), 1,1-dimethylethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)-2,3-dihydro-1H-indole-1-carboxylate (95 g, 275 mmol, 1.25
equiv), Pd2(dba)3 (4.03 g, 4.40 mmol, 0.02 equiv), and K3PO4
monohydrate (101 g, 440 mmol, 2.0 equiv) in dioxane (750 mL)
and water (250 mL) in a 2 L three-neck flask fitted with a condenser
and an overhead stirrer was degassed and backflushed with nitrogen,
followed by addition of tri(tert-butyl)phosphonium tetrafluoroborate
(2.56 g, 8.81 mmol, 0.04 equiv). The mixture was degassed and
backflushed with nitrogen 4 times. The mixture was heated at 85 °C
for 90 min, and the hot mixture was filtered. The hot filtrate was
cooled to room temperature to give a suspension, which was filtered.
The cake was washed with water and dried under house vacuum for 18
h. The cake was then washed with ether and dried to give (53.84 g,
67%) of 1,1-dimethylethyl 5-(4-amino-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-5-yl)-2,3-dihydro-1H-indole-1-carboxylate as a light gray
1
solid. LC−MS (ES) m/z = 366.3 [M + H]+. H NMR (400 MHz,
DMSO-d6) ppm 8.14 (s, 1 H), 7.76 (br s, 1 H), 7.26 (s, 1 H), 7.15−
7.24 (m, 2 H), 6.05 (br s, 2 H), 3.95 (t, J = 8.7 Hz, 2 H), 3.73 (s, 3 H),
3.12 (t, J = 8.6 Hz, 2 H), 1.52 (br s, 9 H).
A mixture of 1,1-dimethylethyl 5-(4-aminofuro[3,2-c]pyridin-3-yl)-
2,3-dihydro-1H-indole-1-carboxylate (1.04 g, 2.96 mmol) and HCl and
4.0 M in dioxane (15 mL, 60.0 mmol) was stirred at room temperature
under nitrogen for 4.5 h. The reaction mixture was then concentrated
in vacuo to give 3-(2,3-dihydro-1H-indol-5-yl)furo[3,2-c]pyridin-4-
amine dihydrochloride (2HCl) (973 mg, 96% yield) as an off-white
solid, which was used without further purification. LC−MS (ES) m/z
= 252 [M + H]+. 1H NMR (400 MHz, DMSO-d6) 8.34 (s, 1 H), 8.06
(d, J = 7.1 Hz, 1 H), 7.60 (br s, 2 H), 7.49 (s, 1 H), 7.36−7.46 (m, 2
H), 7.30 (d, J = 7.8 Hz, 1 H), 4.65 (br s, 2 H), 3.70 (t, J = 8.1 Hz, 2
H), 3.20 (t, J = 8.0 Hz, 2 H).
1,1-Dimethylethyl 5-(4-amino-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-5-yl)-2,3-dihydro-1H-indole-1-carboxylate (3.10 g, 8.48
mmol) was dissolved in a mixture of 2 N HCl (17 mL) and EtOH
(40 mL) as a dark solution. This mixture was filtered through Celite,
washing with 50 mL of 4/1 EtOH/water. The filtrates were heated in
an oil bath at 85 °C (gentle reflux) under nitrogen for 5 h. The clear
brownish solution was cooled to room temperature and concentrated
in vacuo. The slightly wet residue was diluted with 25 mL of EtOH
and heated in a water bath at 65 °C for 5 min. The suspension was
cooled to room temperature. The suspension was filtered, and the cake
was washed with cold EtOH (6 mL) and EtOAc (6 mL). The solids
were dried under vacuum at room temperature for 20 h to give of 5-
(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-
amine·2HCl (2.37 g, 83%) as a light tan powder. LC−MS (ES) m/z =
A mixture of 3-(2,3-dihydro-1H-indol-5-yl)furo[3,2-c]pyridin-4-
amine·2HCl (688 mg, 2.016 mmol), 2,5-difluorophenylacetic acid
(354 mg, 2.057 mmol), HATU (844 mg, 2.220 mmol), and DIPEA
(1.4 mL, 8.02 mmol) in DMF (15 mL) was stirred at room
temperature for 17 h. HPLC indicated complete conversion, so the
mixture was poured into water (75 mL), the suspension was stirred for
about 10 min, and the precipitate was collected by vacuum filtration
and dried by suction to give 3-{1-[(2,5-difluorophenyl)acetyl]-2,3-
dihydro-1H-indol-5-yl}furo[3,2-c]pyridin-4-amine (26) (834 mg, 97%
1
266 [M + H]+. H NMR (400 MHz, DMSO-d6) 3.20 (t, J = 7.8 Hz,
2H), 3.70 (t, J = 8.0 Hz, 2H), 3.85 (s, 3H), 7.27−7.39 (m, 2H), 7.43
(s, 1 H), 7.65 (s, 1 H), 7.77−8.25 (br s, 1 H), 8.53 (s, 1 H).
1
yield) as a tan solid. LC−MS (ES) m/z = 406 [M + H]+. H NMR
To a solution of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-amine·2HCl (200 mg, 0.6 mmol), (2,5-
difluorophenyl)acetic acid (120 mg, 0.696 mmol), and HATU (265
mg, 0.696 mmol) in DMF (5 mL) was added DIPEA (0.463 mL, 2.65
mmol). The mixture was stirred at room temperature overnight. LC−
MS showed the reaction was completed. The mixture was poured into
water. The precipitated solid was filtered and dried to afford 5-{1-
[(2,5-difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (28) as a white solid (208 mg, 74%).
1H NMR showed there is 1 equiv of DMF present. LC−MS (ES) m/z
(400 MHz, DMSO-d6) 3.29 (t, J = 8.34 Hz, 2 H), 3.96 (s, 2 H), 4.31
(t, J = 8.46 Hz, 2 H), 5.52 (s, 2 H), 6.93 (d, J = 5.81 Hz, 1 H), 7.14−
7.34 (m, 4 H), 7.41 (s, 1 H), 7.87 (d, J = 5.81 Hz, 1 H), 7.92 (s, 1 H),
8.13 (d, J = 8.08 Hz, 1 H).
3-{1-[(2,5-Difluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-
1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (27). To a mix-
ture of 5-bromo-1-[(2,5-difluorophenyl)acetyl]-2,3-dihydro-1H-indole
(160 mg, 0.454 mmol), bis(pinacolato)diboron (127 mg, 0.500
mmol), and potassium acetate (134 mg, 1.363 mmol) was added 1,4-
dioxane (6 mL), and the mixture was degassed with N2 for 10 min.
PdCl2(dppf)−CH2Cl2 adduct (18.55 mg, 0.023 mmol) was added, and
the reaction mixture was stirred for 3 h at 100 °C in a sealed vessel.
The mixture was cooled to room temperature. 3-Bromo-1-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (104 mg, 0.454 mmol) and
saturated aqueous NaHCO3 (2 mL) were added, and N2 gas was
bubbled through the mixture for 10 min. PdCl2(dppf)−CH2Cl2 adduct
(18.55 mg, 0.023 mmol) was added. The vessel was sealed, and the
reaction mixture was stirred overnight at 100 °C. The mixture was
= 420 [M + H]+. 1H NMR (400 MHz, DMSO-d6) 3.27 (t, J = 8.46 Hz,
2 H), 3.74 (s, 3 H), 3.95 (s, 2 H), 4.29 (t, J = 8.46 Hz, 2 H), 6.05 (br s,
2 H), 7.21−7.27 (m, 5 H), 7.34 (s, 1 H), 8.09 (d, J = 8.34 Hz, 1 H),
8.15 (s, 1 H).
5-{1-[(2-Fluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (29). To a solution
of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70.6 mg, 0.21 mmol), (2-fluorophenyl)-
acetic acid (32.2 mg, 0.209 mmol), and HATU (79 mg, 0.209 mmol)
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in DMF (2 mL) was added DIPEA (0.15 mL, 0.84 mmol). The
mixture was stirred at room temperature overnight. The mixture was
poured into water, and the precipitated solid was collected and dried
to afford 5-{1-[(2-fluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-
1-Methyl-3-{1-[(4-methylphenyl)acetyl]-2,3-dihydro-1H-
indol-5-yl}-1H-pyrazolo[3,4-d]pyrimidin-4-amine (34). To a
solution of 3-(2,3-dihydro-1H-indol-5-yl)-1-methyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine·2HCl (70 mg, 0.21 mmol), (4-methylphenyl)-
acetic acid (31.0 mg, 0.206 mmol), and HATU (78 mg, 0.206 mmol)
in DMF (2 mL) was added DIPEA (0.144 mL, 0.825 mmol). The
mixture was stirred overnight and then was poured into water. The
precipitated white solid was filtered to give 75 mg (88%) of 1-methyl-
3-{1-[(4-methylphenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (34) LC−MS (ES) m/z = 398.3
[M + H]+. 1H NMR (400 MHz, DMSO-d6) 2.29 (s, 3 H), 3.23 (t, J =
8.46 Hz, 2 H), 3.83 (s, 2 H), 3.93 (s, 3 H), 4.18−4.25 (m, 2 H), 7.14−
7.21 (m, 4 H), 7.44 (dd, J = 8.34, 1.77 Hz, 1 H), 7.50 (s, 1 H), 8.20 (d,
J = 8.34 Hz, 1 H), 8.25 (s, 1 H).
1
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (29) (73 mg, 84%). H
NMR (400 MHz, DMSO-d6) 3.26 (t, J = 8.72 Hz, 2 H), 3.73 (s, 3 H),
3.93 (s, 2 H), 4.28 (t, J = 8.46 Hz, 2 H), 7.19 (d, J = 7.58 Hz, 3 H),
7.26 (s, 1 H), 7.30−7.38 (m, 3 H), 8.09 (d, J = 8.34 Hz, 1 H), 8.14 (s,
1 H). LC−MS (ES) m/z = 402.3 [M + H]+.
5-{1-[(3-Fluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (30). To a solution
of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70.6 mg, 0.21 mmol), (3-fluorophenyl)-
acetic acid (32.2 mg, 0.209 mmol), and HATU (79 mg, 0.209 mmol)
in DMF (2 mL) was added DIPEA (0.146 mL, 0.836 mmol). The
mixture was stirred at room temperature overnight. The mixture was
poured into water, and the precipitated solid was collected and dried
to afford 5-{1-[(3-fluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (30) as a white solid (81
mg, 96%). 1H NMR (400 MHz, DMSO-d6) 3.23 (t, J = 8.46 Hz, 2 H),
3.73 (s, 3 H), 3.92 (s, 2 H), 4.19−4.26 (m, 2 H), 7.08−7.11 (m, 1 H),
7.12−7.17 (m, 2 H), 7.23 (d, J = 8.34 Hz, 1 H), 7.25 (s, 1 H), 7.31 (s,
1 H), 7.36 (s, 1 H), 7.39 (d, J = 6.82 Hz, 1 H), 8.10−8.17 (m, 2 H).
LC−MS (ES) m/z = 402.3 [M + H]+.
5-{1-[(4-Fluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (31). To a solution
of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70 mg, 0.21 mmol), (4-fluorophenyl)acetic
acid (37.5 mg, 0.244 mmol), and HATU (93 mg, 0.244 mmol) in
DMF (2 mL) was added DIPEA (0.162 mL, 0.928 mmol). The
mixture was stirred overnight. The mixture was poured into water (100
mL), and an off-white solid was formed. The solid was filtered, washed
with water (10 mL), and dried to afford 81 mg (84%) of 5-{1-[(4-
fluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-methyl-7H-pyrrolo-
[2,3-d]pyrimidin-4-amine (31) as an off-white solid. LC−MS (ES) m/
z = 402.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6) 3.23 (t, J = 8.34
Hz, 2 H), 3.73 (s, 3 H), 3.88 (s, 2 H), 4.23 (t, J = 8.46 Hz, 2 H), 7.15−
7.20 (m, 2 H), 7.21−7.26 (m, 2 H), 7.30−7.37 (m, 3 H), 8.13 (d, J =
8.34 Hz, 1 H), 8.15 (s, 1 H).
5-{1-[(2-Chlorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (35). To a solution
of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70.6 mg, 0.21 mmol), (2-chlorophenyl)-
acetic acid (39.9 mg, 0.234 mmol), and HATU (89 mg, 0.234 mmol)
in DMF (2 mL) was added DIPEA (0.163 mL, 0.936 mmol). The
mixture was stirred at room temperature overnight, and then the
mixture was poured into water (100 mL), precipitating an off-white
solid. The solid was filtered and dried to afford 94 mg (94%) of 5-{1-
[(2-chlorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (35) as an off-white solid. NMR
indicated 0.8 equiv of DMF was present. LC−MS (ES) m/z = 418.3
[M + H]+. 1H NMR (400 MHz, DMSO-d6) 3.39 (m, 2 H), 3.73 (s, 3
H), 4.00 (s, 2 H), 4.29 (m, 2 H), 7.25 (m, 2 H), 7.30−7.36 (m, 3 H),
7.40 (d, J = 4.55 Hz, 1 H), 7.46 (s, 1 H), 8.09 (s, 1 H), 8.14 (s, 1 H).
5-{1-[(3-Chlorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (36). To a solution
of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70.6 mg, 0.21 mmol), (3-chlorophenyl)-
acetic acid (39.9 mg, 0.234 mmol), and HATU (89 mg, 0.234 mmol)
in DMF (2 mL) was added DIPEA (0.16 mL, 0.94 mmol). The
mixture was stirred at room temperature overnight and then was
poured into water (100 mL). EtOAc (100 mL) was used to extract the
product. The organic phase was dried (MgSO4), filtered, and
evaporated to dryness to give a purple solid that contained some
starting material. The solid was sonicated in water (10 mL), then
filtered and dried to afford 5-{1-[(3-chlorophenyl)acetyl]-2,3-dihydro-
1H-indol-5-yl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine as a
7-Methyl-5-{1-[(2-methylphenyl)acetyl]-2,3-dihydro-1H-
indol-5-yl}-7H-pyrrolo[2,3-d]pyrimidin-4-amine (32). To a sol-
ution of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70.6 mg, 0.21 mmol), (2-methylphenyl)-
acetic acid (31.4 mg, 0.209 mmol), and HATU (79 mg, 0.209 mmol)
in DMF (2 mL) was added DIPEA (0.146 mL, 0.836 mmol). The
mixture was stirred at room temperature overnight. The mixture was
poured into water (100 mL), and a white solid formed. EtOAc (100
mL) was used to extract the product. The organic phase was separated,
dried (MgSO4), and evaporated to give a white solid. The solid was
sonicated in water (10 mL), then filtered and dried to afford 7-methyl-
5-{1-[(2-methylphenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (32) (48 mg, 55%) as a white solid.
1
purple solid. LC−MS (ES) m/z = 418.3 [M + H]+. H NMR (400
MHz, DMSO-d6) 3.24 (t, J = 8.59 Hz, 2 H), 3.73 (s, 3 H), 3.92 (s, 2
H), 4.23 (t, J = 8.46 Hz, 2 H), 6.10 (s, 2 H), 7.23 (d, J = 8.34 Hz, 1 H),
7.26−7.29 (m, 2 H), 7.31−7.33 (m, 1 H), 7.34−7.39 (m, 3 H), 8.12
(d, J = 8.34 Hz, 1 H), 8.15 (s, 1 H).
7-Methyl-5-(1-{[3-(methyloxy)phenyl]acetyl}-2,3-dihydro-
1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (37). To a
solution of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70.6 mg, 0.21 mmol), [3-(methyloxy)-
phenyl]acetic acid (38.9 mg, 0.234 mmol), and HATU (89 mg,
0.234 mmol) in DMF (2 mL) was added DIPEA (0.16 mL, 0.94
mmol). The mixture was stirred at room temperature overnight and
was then poured into water (100 mL), precipitating an off-white solid.
The solid was filtered off and dried to afford 7-methyl-5-(1-{[3-
(methyloxy)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo-
[2,3-d]pyrimidin-4-amine as an off-white solid (91 mg, 90%). LC−MS
(ES) m/z = 414.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6) 3.21 (t,
J = 8.46 Hz, 2 H), 3.73 (s, 3H), 3.75 (s, 3H), 3.84 (s, 2 H), 4.20 (t, J =
8.46 Hz, 2 H), 6.06 (br s, 2 H), 6.82−6.90 (m, 3 H), 7.21−7.26 (m, 3
H), 7.28−7.31 (m, 1 H), 8.11−8.19 (m, 1 H), 8.14 (s, 1 H).
7-Methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihy-
dro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (38).
To a stirred suspension of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine·2HCl (20 0.0 g, 59.1 mmol, 1
equiv) and HATU (24.73 g, 65.0 mmol, 1.1 equiv) in DMF (160 mL)
chilled in an ice bath was added DIEA (33.0 mL, 189 mmol, 3.2 equiv)
in one portion. The mixture turned into a clear light brown solution.
To this mixture was added [3-(trifluoromethyl)phenyl]acetic acid
1
LC−MS (ES) m/z = 398.3 [M + H]+. H NMR (400 MHz, DMSO-
d6) 8.02−8.24 (m, 2 H), 7.32 (s, 1 H), 7.25 (s, 1 H), 7.12−7.24 (m, 5
H), 6.07 (br s, 2 H), 4.26 (t, J = 8.5 Hz, 2 H), 3.87 (s, 2 H), 3.73 (s, 3
H), 3.24 (t, J = 8.5 Hz, 2 H), 2.24 (s, 3 H).
7-Methyl-5-{1-[(3-methylphenyl)acetyl]-2,3-dihydro-1H-
indol-5-yl}-7H-pyrrolo[2,3-d]pyrimidin-4-amine (33). To a sol-
ution of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine·2HCl (70.6 mg, 0.21 mmol), (3-methylphenyl)-
acetic acid (31.4 mg, 0.209 mmol), and HATU (79 mg, 0.209 mmol)
in DMF (2 mL) was added DIPEA (0.146 mL, 0.836 mmol). The
mixture was stirred at room temperature overnight. The mixture was
poured into water (100 mL), and a light brown solid formed. The solid
was filtered and dried to afford 7-methyl-5-{1-[(3-methylphenyl)-
acetyl]-2,3-dihydro-1H-indol-5-yl}-7H-pyrrolo[2,3-d]pyrimidin-4-
1
amine (33) (48 mg, 55%). LC−MS (ES) m/z = 398.3 [M + H]+. H
NMR (400 MHz, DMSO-d6) 2.30 (s, 3 H), 3.16−3.23 (m, 2 H), 3.72
(s, 3 H), 3.82 (s, 2 H), 4.17−4.24 (m, 2 H), 7.06−7.14 (m, 3 H),
7.20−7.27 (m, 3 H), 7.30 (s, 1 H), 8.11−8.18 (m, 2 H).
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(12.07 g, 59.1 mmol, 1 equiv) portionwise (2 g per 15 min) over a 2 h
period. The cooling bath was removed upon completion of the
addition. The mixture was stirred at ambient temperature for another
2.5 h and then poured into 1 L of ice cold water to form a suspension,
which was filtered. The cake was washed with water and dried under
house vacuum for 1.5 h. The solid was dissolved in 500 mL of 10%
MeOH in CH2Cl2, dried over Na2SO4, filtered, and concentrated in
vacuo to give the crude product (39.5 g) as a brown foam. The crude
product was dissolved in 100 mL of CHCl3, split into 12 equal
portions, and chomatographed on silica gel cartridges (Analogix SF40-
115g) using a gradient elution of 1% A to 60% A in CHCl3 (A was a
mixture of 3200/800/80 CHCl3/MeOH/NH4OH). Impure product
fractions were combined and chromatographed using a gradient
elution of 1% B in EtOAc to 75% B in EtOAc (B was a mixture of 20%
MeOH in EtOAc). The combined purified product was taken up in
CHCl3 (23 mL) and MTBE (140 mL) as a slurry, which was filtered.
The cake was washed with MTBE (2 × 100 mL) and hexane (3 × 75
mL). The solids were then dried under vacuum at 65 °C for 48 h to
afford 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-
1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (38) (20.64 g,
75%) as a white solid. LC−MS (ES) m/z = 452 [M + H]+. 1H
NMR (400 MHz, DMSO-d6) 3.25 (t, J = 8.3 Hz, 12 H), 3.73 (s, 13
H), 4.03 (s, 2 H), 4.27 (t, J = 8.5 Hz, 2 H), 7.18−7.28 (m, 2 H), 7.32
(s, 1 H), 7.55−7.67 (m, 3 H), 7.68 (s, 1 H), 8.07−8.14 (m, 1 H), 8.15
(s, 1 H). Anal. Calcd for C24H20F3N5O: 63.85% C, 4.47% H, 15.69%
N. Found: 63.82% C, 4.50% H, 15.70% N.
5-(1-{[3-Fluoro-5-(trifluoromethyl)phenyl]acetyl}-2,3-dihy-
dro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (39). A solution of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine 2HCl (150 mg, 0.443 mmol), [3-
fluoro-5-(trifluoromethyl)phenyl]acetic acid (99 mg, 0.443 mmol),
HATU (169 mg, 0.443 mmol), and DIEA (0.310 mL, 1.774 mmol)
was stirred at room temperature overnight. The reaction mixture was
poured into water (10 mL), and a precipitate formed. The precipitate
was collected by filtration, and the residue was washed with water (10
mL) and dried. The beige solid was adsorbed onto silica and purified
by flash chromatography (0−10% methanol in CH2Cl2, 12 g silica
column) to afford a pale yellow solid which showed the presence of
bis-acylated material. The product was adsorbed onto silica and
purified by flash chromatography (100% EtOAc to 10% MeOH in
EtOAc, then 10% MeOH in CH2Cl2, 12 g column) to afford 5-(1-{[3-
fluoro-5-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-
7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (39) (110 mg, 53%
yield) as a white solid. LC−MS (ES) m/z = 470 [M + H]+. 1H NMR
(400 MHz, DMSO-d6) 3.27 (t, J = 8.34 Hz, 2 H), 3.74 (s, 3 H), 4.07
(s, 2 H), 4.27 (t, J = 8.46 Hz, 2 H), 5.81−6.32 (m, 2 H), 7.17−7.29
(m, 2 H), 7.33 (s, 1 H), 7.52 (d, J = 9.60 Hz, 1 H), 7.55−7.63 (m, 2
H), 8.11 (d, J = 8.34 Hz, 1 H), 8.15 (s, 1 H).
7-Methyl-5-{1-[(2,3,5-trifluorophenyl)acetyl]-2,3-dihydro-
1H-indol-5-yl}-7H-pyrrolo[2,3-d]pyrimidin-4-amine (40). A sol-
ution of 5-(2,3-dihydro-1H-indol-5-yl)-7-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine (150 mg, 0.443 mmol), (2,3,5-trifluorphenyl)acetic
acid (84 mg, 0.443 mmol), HATU (169 mg, 0.443 mmol), and DIEA
(0.310 mL, 1.774 mmol) was stirred at room temperature overnight.
The reaction mixture was poured into water (10 mL), and a precipitate
formed. The precipitate was collected by filtration, washed with water
(10 mL), and dried. The solid was adsorbed onto silica and purified by
flash chromatography (0−10% methanol in DCM, 12-g column) to
afford a pale yellow solid which showed the presence of bis-acylated
material. The product was adsorbed onto silica and purified by flash
chromatography (100% EtOAc to 10% MeOH in EtOAc, then 10%
MeOH in CH2Cl2, 12 g column) to afford 7-methyl-5-{1-[(2,3,5-
trifluorophenyl)acetyl]-2,3-dihydro-1H-indol-5-yl}-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine (40) (102 mg, 53% yield) as a white solid. LC−MS
(ES) m/z = 438 [M + H]+. 1H NMR (DMSO-d6, 400 MHz) 3.74 (s, 3
H), 4.04 (s, 2 H), 4.29 (t, J = 8.5 Hz, 2 H), 6.08 (br s, 2 H), 7.10−7.17
(m, 1 H), 7.20−7.28 (m, 2 H), 7.34 (s, 1 H), 7.42−7.53 (m, 1 H), 8.08
(d, J = 8.1 Hz, 1 H), 8.15 (s, 1 H). 3.28 (t, J = 8.3 Hz, 2 H).
ASSOCIATED CONTENT
* Supporting Information
Complete kinase profiling data for compound 38 and cloning,
expression, purification, and crystallization details for human
PERK with compounds 22 and 38. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
Accession Codes
Protein Data Bank submissions codes: compound 22, 4G34;
compound 38, 4G31.
AUTHOR INFORMATION
Corresponding Author
*Telephone: 610-917-7899. E-mail: Jeffrey.M.Axten@gsk.com.
Notes
■
The authors declare no competing financial interest.
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