Design, Synthesis, and Biological Evaluation of Novel Indoles Targeting the Influenza PB2 Cap Binding Region

Article abstract of DOI:10.1021/acs.jmedchem.9b01091

In the search for novel influenza inhibitors we evaluated 7-fluoro-substituted indoles as bioisosteric replacements for the 7-azaindole scaffold of Pimodivir, a PB2 (polymerase basic protein 2) inhibitor currently in clinical development. Specifically, a 5,7-difluoroindole derivative 11a was identified as a potent and metabolically stable influenza inhibitor. 11a demonstrated a favorable oral pharmacokinetic profile and in vivo efficacy in mice. In addition, it was found that 11a was not at risk of metabolism via aldehyde oxidase, an advantage over previously described inhibitors of this class. The crystal structure of 11a bound to influenza A PB2 cap region is disclosed here and deposited to the PDB.

Full text of DOI:10.1021/acs.jmedchem.9b01091

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Article
Design, Synthesis, and Biological Evaluation of Novel
Indoles Targeting the Influenza PB2 Cap Binding Region
David Craig McGowan, Wendy Balemans, Werner Embrechts, Magali Motte, Jeremy Keown,
Christophe Buyck, Jordi Corbera, Mario Funes, Laura Moreno, Ludwig Cooymans, Abdellah Tahri,
Julien Eymard, Bart Stoops, Rudy Strijbos, Joke Van den Berg, Ervin Fodor, Jonathan Grimes,
Anil Koul, Tim H.M. Jonckers, Pierre Jean Marie Bernard Raboisson, and Jerome Guillemont
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01091 • Publication Date (Web): 24 Oct 2019
Downloaded from pubs.acs.org on October 25, 2019
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Design, Synthesis, and Biological Evaluation of Novel Indoles
Targeting the Influenza PB2 Cap Binding Region
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†,*
†
†
§
David C. McGowan, Wendy Balemans, Werner Embrechts, Magali Motte, Jeremy
⏀
†
‡
‡
‡
R. Keown, Christophe Buyck, Jordi Corbera, Mario Funes, Laura Moreno, Ludwig
†
†

†
°
Cooymans, Abdellah Tahri, Julien Eymard, Bart Stoops, Rudy Strijbos, Joke Van
†
∞
⏀,◊
†
†
den Berg, Ervin Fodor, Jonathan M. Grimes, Anil Koul, Tim H.M. Jonckers, Pierre
†
§
Raboisson, Jérôme Guillemont
†Janssen Pharmaceutica, N.V. Turnhoutseweg 30, 2340 Beerse, Belgium
§Janssen-Cilag, Campus de Maigremont BP615, F-27106 Val de Reuil Cedex, France
Novalix (site Janssen-Cilag), Campus de Maigremont BP615, F-27106 Val de Reuil Cedex,
France
⏀Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of
Oxford, Oxford OX3 7BN, UK
∞Sir William Dunn School of Pathology, University of Oxford, South Parks Rd, Oxford OX1 3RE,
UK
◊Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, UK
‡Eurofins Villapharma Research, S.L., Avda. Isaac Peral, Parque Tecnológico de Fuente Álamo,
Ctra. El Estrecho-Lobosillo, 30320 Murcia, Spain
°Charles River Labs Beerse, Turnhoutseweg 30, 2340 Beerse, Belgium
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KEYWORDS. Influenza, indole, bioisostere
Abstract. In the search for novel influenza inhibitors we evaluated 7-fluoro-substituted
indoles as bioisosteric replacements for the 7-azaindole scaffold of Pimodivir, a PB2
(polymerase basic protein 2) inhibitor currently in clinical development. Specifically, a 5,7-
difluoroindole derivative 11a was identified as a potent and metabolically stable influenza
inhibitor. 11a demonstrated a favorable oral pharmacokinetic profile and in vivo efficacy
in mice. In addition, it was found that 11a was not at risk of metabolism via aldehyde
oxidase, an advantage over previously described inhibitors of this class. The crystal
structure of 11a bound to influenza A PB2 cap region is disclosed here and deposited to
the PDB.
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INTRODUCTION
Influenza is an infectious disease affecting the upper respiratory tract and often the
lungs and is caused by the influenza virus. Recent data show that every year, 3 to 11%
of the US population become infected with influenza virus and some require
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hospitalization. The ability of influenza to be transmitted from animals to human makes
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it particularly dangerous and has led to global pandemics. While vaccination is
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recommended to reduce infection rates and decrease the likelihood of an epidemic, it is
not efficacious for everyone, with 50-60% effectiveness at reducing the infection risk in
the global population, and nearly 60% effective at reducing morbidity in the elderly. This
may be due to the way the vaccine is designed (i.e. live attenuated vs. inactivated
influenza vaccines), the relatively low vaccination rates in some countries, or that the
predominant seasonal viral strain has evaded the host immune system due to
antigenetic drift.1, 3
O
O
O
OH
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OH
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NH
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N
H
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HN
NH2
OH
F
N
H
N
S
H
NH2
NH
Zanamivir (2)
F
Oseltamivir (1)
Xofluza (3)
Pimodivir (4)
Figure 1. Selected anti-influenza agents.
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Among the classes of approved influenza inhibitors are the neuraminidase inhibitors, that
are the current standard of care. Oseltamivir (1) is given orally, and Zanamivir (2) is
marketed for inhalation. Recently, Xofluza (3) was approved as a first-in-class drug with
a novel mechanism of action targeting the viral cap-dependent endonuclease. It is
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reported to be effective against Oseltamivir-resistant strains. These agents are highly
effective against influenza types A and B but share a common drawback, as they must
be administered within the first few days of infection, and preferentially before the onset
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of symptoms to make meaningful therapeutic impact. Furthermore, resistance has been
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7
shown to occur for neuraminidase and endonuclease inhibitors. Thus, there is an
immediate medical need for new drugs to cover novel mechanisms of action that could
8
be used seasonally or to help prevent a future pandemic. Pimodivir (4) is described as a
9
PB2 cap binder, and is currently in clinical development for the treatment of severe
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influenza infection in high risk and hospitalized patients. PB2, a subunit of the influenza
virus RNA polymerase complex, plays an essential role in transcription initiation and the
cap-stealing mechanism, and acts as a virulence determinant.11
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MEDICINAL CHEMISTRY STRATEGY
The recent publication of the crystal structure of 4 bound to the cap region has led
research groups to pursue new influenza inhibitors. One strategy of exploring new
inhibitors is to systematically explore each ring system of 4 and replace selected groups
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by moieties having similar biological properties, termed bioisosteres, that could modify
the activity or reduce off-target activity. The design of bioisosteric replacements is a
valuable strategy to develop alternative drug candidates in a scaffold-hopping exercise
13
to potentially address aspects of lead candidates. We envisioned that the fluoroindole
could be an effective bioisostere of the 7-azaindole ring system with differentiated
properties (e.g. polar surface area and calculated logP) and would have the potential to
find application in other targets (e.g. nucleoside bases). Indeed, bioisosteres of 4 have
14
been described wherein the acid motif was replaced by a heterocycle, also the central
pyrimidine ring of 4 was bioisosterically exchanged for pyrimidine-fused heterocycles.15
This work covers the first described bioisostere of 7-azaindole, resulting in an influenza
inhibitor that is effective in vivo and has reduced off-target activity.
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Examining the structure of 4, we reasoned that a carbon-fluorine bond could act as
16
hydrogen bond acceptor. Evidence of a similar scaffold-hopping exercise was
described in the literature where 7-fluoro-7-deazaadenosine-nucleotide was
incorporated as efficiently as adenosine into T7 RNA polymerase, despite lacking the
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nitrogen and possessing a different pKa value. This intriguing fact provides rationale
for new possibilities in medicinal chemistry scaffold design, in that the small fluorine
group can have minimal structural influence, yet imitate an aromatic nitrogen atom,
allowing the molecule to adopt a similar binding mode.
CHEMISTRY
The first Pimodivir analog of interest was the 5,7-difluoroindole bioisostere. In addition,
other substituents in the indole-5-position that were of similar size or electronic
character were selected for comparison (e.g. 5-CH , vide infra).
3
The synthesis towards the first example, 5,7-difluoroindole analogue 11a, began with
commercially available 5,7-difluoroindole (5a) as shown in Scheme 1. The indole
nitrogen was tosyl protected in phase transfer catalyst conditions, resulting in 6a in high
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yield, which facilitated subsequent bromination with NBS in dichloromethane (7a). The
aryl bromide was then reacted with bis(pinacolato)diboron under standard conditions to
afford boronic ester 8a in modest yield (52%). The pyrimidine bicyclo[2.2.2]octane
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aminoester (9) was prepared according to literature, and coupled with 8a under
standard Suzuki-Miyaura reaction conditions. Hydrolysis of the ester using lithium
hydroxide in a dioxane and water mixture afforded 11a, the first example bioisostere of
4. The corresponding 5-chloro-7-azaindole analogue was described as potent and cell
10
penetrant, and was thus also prepared. In addition, compounds 5c and 5d were
prepared as the methyl group shares the same relative size as the chlorine, and a nitrile
would have electron withdrawing properties like that of fluorine.18
The synthesis varied slightly for each of the remaining targeted indole influenza inhibitors.
For example, in the tosyl protection step to form 6c, NaH in DMF was used in the absence
of a phase transfer catalyst in high yield (92%). Another variation was in the bromination
step to form 3-bromo-5-cyano-7-fluoro-1-tosyl-1H-indole (7d) where molecular bromine
was employed in dichloromethane. Trivial disparities in the Miyaura borylation and
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subsequent Suzuki-Miyaura reactions are detailed in the experimental section. Finally, in
the last step, the base catalyzed hydrolysis to form the final indole inhibitors 11a-d, had
some differences in the choice of base and means of purification and resulted in a wide
range of yields (7 to 79%).
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Scheme 1. General synthetic route toward (2S,3S)-3-((2-(5-R-7-fluoro-1H-indol-3-yl)-5-
fluoropyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acids (11a-d).
O
R
F
O
X
B
N
R
R
R
V
VI
F
NH
O
I or II
N
Y
N
N
O
N
H
N
F
N
Ts
Ts
O
F
F
NH
H
F
Cl
N
O
Entry, R
5a, F
6a-d, X = H
7a-d, X = Br
8a-d
9
10a-d, Y = CH3
11a-d, Y = H
III or IV
VII - IX
5b, Cl
5c, CH3
5d, CN
Reagents and conditions: I) TsCl, NaOH/toluene, TBAHS, rt, 2-3h, 63-91% or II) TsCl, NaH, DMF, 0ºC to
rt, 2h, 92% III) NBS, CH Cl , rt, 18h, 68 - 93%, or IV) Br , CH Cl , 0°C to rt over 1.5h, 76% V)
bis(pinacolato)diboron, PdCl (dppf), KOAc, dioxane, 90°C for 18h, 45-96% VI) PdCl (dppf), K PO
dioxane, water, microwave 100°C, 32-68% VII) For 11a,b; LiOH, dioxane, water, rt, 18h, 71- 79% VIII) For
1c; KOH, EtOH, 80°C, 40 min, 7% IX) For 11d; NaOCH , CH OH, rt for 18h, 64%.
2
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,
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RESULTS AND DISCUSSION
The cellular antiviral activity of 11a-d were tested against three influenza A strains, two
H1N1 strains and one H3N2 strain, and cytotoxicity was measured in MDCK and A549
cells (Table 1). All tested analogues had a cellular potency in the low nM range
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(
Oseltamivir and Favipiravir were used as reference molecules). Higher selectivity
indices (CC /IC ) were observed in the A549 cell line (derived from human alveolar
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epithelium) compared to MDCK cells (derived from madin-darby canine kidney), but
comparable for the four analogues within each cell line.
Table 1. Antiviral activity of indole variations against three influenza A virus strains and
cytotoxicity in MDCK and A549 cells.
R
F
N
O
NH
N
OH
F
N
H
a,c
a,c
a,d
A/Taiwan/1/86
A/Virginia/3/1975
A/Puerto Rico/8/34
Compound
1a
R
b
SIe
145
83
75
97
b
SIe
114
28
25
59
b
SIe
IC , nM
IC , nM
IC , nM
5
0
50
50
1
F
Cl
11
21
4
11
16,800
14
47
11
19
35,700
22
53
22
43
31
>1142
>485
1491
>612
>4132
1
1b
1c
11d
1
CH3
CN
f
Reference
>6
>3
a
b
c
d
e
f
A/Taiwan/1/86 and A/Puerto Rico/8/34 are H1N1 strains, A/Virginia/3/1975 is a H3N2 strain.
IC50 is the 50% inhibiting concentration, i.e. the concentration causing 50% decrease of viral replication.
MDCK cells.
A549 cells.
SI selectivity index, CC50/IC50
.
Reference molecules used: Favipiravir in MDCK cells, and Oseltamivir in A549 cells.
The biochemical binding affinity of the analogues to the PB2 cap binding domain of
®
influenza A and B was assessed in a Lance Ultra competitive binding assay (Table 2).
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In this assay, influenza A affinities were comparable for the tested analogues, and a trend
was observed towards influenza B affinity, albeit in µM range, for 11c and 11d.
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Table 2. Inhibition of CAP-Binding (γ-[6-aminohexyl]-GTP-biotin ligand) to A/Victoria/3/1975 and
B/Brisbane/60/2008 PB2CAP-binding domains.a
A/Victoria/3/1975
B/Brisbane/60/2008
Compound a
b
b
IC , µM
IC , µM
50
50
11a
0.12
0.17
0.12
0.08
>50
>50
26
1
1b
1c
1d
1
1
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a
All compounds have been tested in the Protein L Lance Ultra counter screen that assessed non-specific technology binders. IC50
values of >50 µM were obtained for all compounds. A/Victoria/3/1975 is an H3N2 strain.
IC50 is the 50% inhibiting concentration, i.e. the concentration causing 50% decrease in CAP-binding.
b
Figure 2. Superposition of 4 (dark green) and 11a (orange). The binding pocket is shown as a surface. The
hydrogen bonding network formed between the protein (with Lys376 and Glu361, highlighted residues in
bright yellow) and the ligands is shown as dotted lines between the ligand and the protein residues, the CF-
lysine interaction of 11a is very similar to the N-lysine interaction of 4.
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Initially, without access to a crystal structure, a docking experiment was done, using the
previously reported crystal structure (PDB ID: 5WL0), to better understand the role of the
1
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7-fluoroindole and the influence on the binding mode (Fig. 2). The distance between the
fluorine atom in the 7-position of the 5,7-difluoroindole of 11a and the terminal nitrogen
atom of the amino group of Lys376 is 2.5 Å with an angle of 156°. As a comparison, the
related 7-azaindole nitrogen of 4 and the terminal amino group of Lys376 distance is 3.4
Å. The fact that C-F groups can act as weak H-bond acceptors,16,19 and given the close
proximity of 2.5 Å, it is conceivable that there is a weak hydrogen bond or dipole
interaction occuring. Indeed, similar configurations in the PDB are observed where the
fluorine on an aromatic ring is in proximity of less than 3.2 Å to the terminal amino group
of lysine side chain having N-F-C angles that ranged from 70° to 160° (Table S1). The
docking and the presence of formerly crystalized ligands in a similar environment
supports the rationale of the potential use of fluorine on aromatic rings as a hydrogen
bond acceptor and allows us to use the model to design new compounds.
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Structure analysis by X-ray diffraction was performed to obtain a more definitive
assessment of the binding mode. The best crystal diffracted to a resolution of 1.35 Å and
was refined to Rwork/Rfree values of 14.7%/16.5%. The two molecules in the asymmetric
are essentially identical with an RMSD of 0.23 over all Cα-atoms. The cap-binding domain
is in the “transcriptionally inactive” conformation similar to that seen for the influenza C
polymerase heterotrimer in the absence of viral RNA and previously determined
structures of the cap midlink domains with cap analogues (Fig.3A).20-22
0
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A very clear electron density was observed for 11a and was placed unambiguously (Fig
3B). Interestingly, though unlikely to be biologically relevant, an additional molecule of
11a that mediates crystal contacts was observed. The cap-binding domain provides many
of the residues that interact with compound 11a. The indole moiety of 11a forms -
stacking interactions residues Phe404 and His357, as well as ionic interactions with
Glu361 and Lys376. Additional interactions with 11a from the cap-binding domain are
provided by Phe323 that forms  -stacking with the pyrimidine ring, and Arg355 that forms
two ionic interactions with the carboxylate of 11a. Consistent with previous observations
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an ionic interaction between the backbone carboxyl of Val511 from the 627-link domain
with the amine at the 4-position of the pyrimidine of 11a was observed.22
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Comparison to the previously determined structures of 4 in complex with a similar cap-
midlink construct to that used in this study (PBD ID: 6EUV) shows a globally similar
structure with local points of difference (Fig. 3 C/D). At the purine end of the molecule,
where the 7-azaindole nitrogen from 4 is substituted with C-F in 11a, Lys376 that
previously interacted with the nitrogen now forms an interaction with the fluorine group.
The change in this interaction pushes 11a approximately 0.8 Å out of the pocket. The
movement out of the pocket facilitates a rotation of the carboxylate group 90° such that it can now
form two ionic interactions with Arg355 rather than one. The global outcome of the new binding
position of 11a is that the cap binding site, created between the cap binding and mid/627-
link domains is in a more open conformation, consistent with the predicted mode of
binding.
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Figure 3. A) Domain arrangement of the mid (dark red), cap-binding (gold), and 627-link domains
(green). B) Detail of binding of 11a with important residues and interaction distances for key
residues (PDB ID: 6S5V). Electron density is contoured at 3σ. C) Opening of the binding site in
the presence of 4 (purple) (PDB ID: 6EUV) and 11a (colored as per panel A). D) Detailed
interactions for the binding of 11a vs 4 (distances shown for 4).
A comparison of the in vitro metabolic stability of the four inhibitors showed that the 5,7-
difluoroindole 11a had superior stability in human liver microsomes (Cl < 7.7 µL/min/mg
int
protein). In addition, 11a was found to be a crystalline solid as a free base, an
advantageous property for further development. 11a was found to be inactive across a
panel of 59 kinases, including GSK3 (no inhibition at 1M), had no toxicity in the AMES
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II test, and permitted no formation of micronuclei in TK-6 cells. As an early cardiovascular
safety assessment, 11a showed a lack of significant binding to sodium or potassium
hERG channels (> 10 µM). A differentiating factor was that 11a does not show aldehyde
oxidase (AO) metabolism in human liver cytosol (1h, 1 M), contrary to the previously
reported 7-azaindole inhibitors.14,23
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The pharmacokinetic profile of 11a in mice was investigated based on the encouraging
potent in vitro inhibition of influenza and confirmed target engagement in the PB2 cap
binding region. The mouse plasma protein binding was high, and similar to human (99.92
%). 11a was administered orally to healthy balb/c mice at a dose of 10 mg/kg. The
compound was found to be rapidly absorbed with a Tmax between 0.5 and 2h and
demonstrated good oral bioavailability (Table 4). A satellite group (n = 3) was dosed p.o.
to determine the lung and plasma concentrations of 11a at 7h. Mean plasma
concentration was 1005 ng/mL and mean lung concentration was 491 ng/g affording a
lung/plasma ratio of approximately 0.5. Higher clearance was observed in dogs despite
greater liver microsomal stability, nevertheless bioavailability remained favorable.
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Table 4. Mean Pharmacokinetic parameters following single oral dose of 11a in mice and dogs.*
Clint, Microsomes
C
max
AUClast
Species Dose PPB
in vitro
T½ (h)
F (%)
(
ng/mL)
(hr*ng/mL)
(µL/min/mg protein)
Mouse
Dog
10
99.90
99.94
15
3490 ± 1200 16100 ± 2420
1090+/-505 3020+/-719
3.6
5.8
95 ± 14
68 ± 11
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<7.7
*
PEG400 was used as the vehicle.
Compound 11a was consequently selected for evaluation in a mouse lethal infection
model induced by influenza A/Puerto Rico/8/34 (H1N1) using female balb/c mice.
Therapeutic efficacy, with 75% survival observed at day 21 post infection, was
demonstrated when the compound was administered orally for 10 days twice daily at 30
mg/kg starting 48 hours post infection. (Fig. 4). In addition, an initial significant body
weight loss was observed in this treatment setting due to the infection, however, the
animals regained weight from day 11 and increased onwards (Figure S2). Tamiflu was
included as a reference compound, where mice showed 38% survival at day 21 post
infection in a therapeutic setting (10 mg/kg oral b.i.d. treatment for 10 days starting 48
hours post infection) (Figure 4).
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Days post infection
Figure 4. Mouse in vivo efficacy of 11a administered orally (30 mg/kg) 48h post infection for 10
days BID to female balb/c mice (n=8/group) inoculated with influenza A/Puerto Rico/8/34 (blue).
Tamiflu (10 mg/kg orally BID; treatment start 48 hours post infection and for 10 days) was included as a
reference compound (red). Vehicle-treated group (PEG400) is shown in black. See figure S2, body weight
as percent change from baseline, in the supporting information.
CONCLUSIONS
In conclusion, we have identified 7-fluoroindole as a bioisostere of 7-azaindole. This novel
class of indole-based influenza inhibitors demonstrated target engagement by binding to
influenza PB2 in the binding assay as well as potent inhibition of virally infected cells. In
particular, the more metabolically stable 5,7-difluoroindole analog 11a demonstrated
value as a bioisostere for the 7-azaindole in 4 and was effective as an influenza PB2
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inhibitor. Oral administration of 11a demonstrated high exposure in mice and dogs,
distribution to target tissue, and in vivo efficacy was shown in a mouse lethal infection
model. The benefit of the 7-fluoroindole also likely led to the reduced kinase activity and
the dearth of aldehyde oxidase metabolism.
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EXPERIMENTAL SECTION
Chemistry. Reagents and solvents were purchased from commercial sources and used
without further purification. NMR spectra were recorded on a Bruker 400, 500, or 600 MHz
spectrometer. Chemical shifts and multiplicity data are given according to the ACS NMR
®
guidelines. High resolution mass spectrometry was performed on a Waters Acquity IClass
®
UPLC -DAD and Xevo G2-S QTOF. The samples were run on a Waters BEH C18 (1.7 µm, 2.1
x 50 mm, at 50ºC) column using reverse phase chromatography with a gradient from 95% A to
5
% A in 4.6 minutes, held for 0.4 minutes (A: 95% 6.5 mM CH COONH / 5% CH CN, B:
3 4 3
CH CN). All compounds have purity greater than 97%.
3
5,7-difluoro-1-tosyl-1H-indole (6a). 5,7-difluoroindole (500 mg, 3.27 mmol) was added
to toluene (8 mL) while stirring under nitrogen. Tetrabutylammonium hydrogen sulfate (83 mg,
0
.25 mmol) was added followed by NaOH (50% in H O, 5 mL) and the mixture was stirred
2
vigorously. A solution of p-toluenesulfonyl chloride (654 mg, 3.43 mmol) in toluene (8 mL) was
added and the mixture was stirred for 3h at room temperature. The organic layer was separated
and washed with water, dried over MgSO , the solids were removed by filtration, and the filtrate
4
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was concentrated under reduced pressure to afford the titled compound as a white solid (0.9 g,
1
88%). H NMR (400 MHz, DMSO-d ) δ 7.98 (d, J=3.52 Hz, 1H), 7.79 (d, J=7.70 Hz, 2H), 7.43
6
(d, J=7.72 Hz, 2H), 7.33 (dd, J=2.31, 8.47 Hz, 1H), 7.17 (ddd, J=2.20, 9.68, 12.10 Hz, 1H), 6.91
1
1
1
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+
(dd, J=2.31, 3.63 Hz, 1H), 2.35 (s, 3H). MS (ESI) m/z: 325.1 [M+NH ] (calcd for
4
C H F N O S: 325.08).
15 15
2
2
2
5-chloro-7-fluoro-1-tosyl-1H-indole (6b). The titled compound was prepared according
1
to the method to prepare 5,7-difluoro-1-tosyl-1H-indole, (63%). H NMR (400 MHz, CDCl ) δ
3
7
.76-7.82 (m, 3H), 7.24-7.31 (m, 3H), 6.95 (dd, J=1.76, 11.44 Hz, 1H), 6.60 (dd, J=2.20, 3.74
+
Hz, 1H), 2.37 (s, 3H). MS (ESI) m/z 324.2 [M+H] (calcd for C H ClFNO S: 324.03).
1
5
12
2
7
-fluoro-5-methyl-1-tosyl-1H-indole (6c). NaH (60% dispersion in mineral oil) (0.87 g,
21.72 mmol) was added portionwise to a solution of 7-fluoro-5-methyl-1H-indole (2.7 g, 18.1
mmol) in DMF (30 mL) at 0°C purged with N flow, and then the mixture was stirred for 30 min
2
at 0°C. p-Toluenesulfonyl chloride (3.62 g, 19.01 mmol) was added portion wise at 0°C then the
reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was poured
into ice water (90 mL) and stirred for 10 min. EtOAc was added and the organic layer was
extracted, dried over MgSO , the solids were removed by filtration, and the solvent of the filtrate
4
1
was removed under reduced pressure to afford the titled compound (5.08 g, 92%). H NMR (500
MHz, DMSO-d ) δ ppm 7.84 (d, J=3.8 Hz, 1 H) 7.76 (d, J=8.2 Hz, 2 H) 7.41 (d, J=8.2 Hz, 2 H)
6
7
.22 (s, 1 H) 6.9 (d, 1 H, J=13.2 Hz) 6.82 (dd, J=3.6, 2.4 Hz, 1 H) 2.33 (s, 3 H) 2.31 (s, 3 H). MS
+
(
ESI) m/z: 321.3 [M+NH ] (calcd for C H FN O S: 321.11).
4
16 18
2
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-fluoro-1-tosyl-1H-indole-5-carbonitrile (6d). 7-fluoro-1H-indole-5-carbonitrile (1.90
g, 11.86 mmol) was added to toluene (10 mL) while stirring under nitrogen. Tetrabutylammonium
hydrogen sulfate (402 mg, 1.19 mmol) was added followed by NaOH (50% in H O, 10 mL) and
2
0
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the mixture was stirred vigorously. A solution of p-toluenesulfonyl chloride (3.39 g, 17.80 mmol)
in toluene (8 mL) was added and the mixture was stirred for 16h. The organic layer was separated
and washed with water, dried over MgSO , the solids were removed by filtration and the solvent
4
of the filtrate was concentrated under reduced pressure. The crude was purified by silica column
chromatography using a heptane to EtOAc gradient. The desired fractions were collected and
1
evaporated to dryness to afford 7-fluoro-1-tosyl-1H-indole-5-carbonitrile (3.4 g, 91%). H NMR
(
400 MHz, DMSO-d ) δ 8.12 (d, J=3.74 Hz, 1H), 8.08 (d, J=1.32 Hz, 1H), 7.83-7.90 (m, 2H), 7.71
6
(
dd, J=1.10, 12.10 Hz, 1H), 7.47 (d, J=8.14 Hz, 2H), 7.04 (dd, J=2.20, 3.74 Hz, 1H), 2.37 (s, 3H).
-
MS (ESI) m/z: 313.2 [M-H] (calcd for C H FN O S: 313.04)
1
6
12
2
2
3
-bromo-5,7-difluoro-1-tosyl-1H-indole (7a). N-bromosuccinimide (9.64 g, 54.18
mmol) was added portion wise to a solution of 5,7-difluoro-1-tosyl-indole (16.65 g, 54.18 mmol)
in CH Cl (300 mL) at room temperature and stirred for 18h. The mixture was treated with a
2
2
saturated aq. NaHCO solution and the mixture was stirred for 5 min. The organic layer was dried
3
(
MgSO ), the solids were removed by filtration and the solvent was removed under reduced
4
pressure. The crude was purified by silica column chromatography using a heptane to CH Cl
2
2
gradient. The desired fractions were collected and evaporated to dryness to afford the titled
1
compound as a tan solid (0.9 g, 79%). H NMR (400 MHz, DMSO-d ) δ 8.27 (s, 1H), 7.86 (d,
6
J=7.70 Hz, 2H), 7.45 (d, J=7.92 Hz, 2H), 7.34 (ddd, J=2.31, 9.63, 12.05 Hz, 1H), 7.18 (dd, J=1.98,
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+
7.92 Hz, 1H), 2.36 (s, 3H). MS (ESI) m/z: 409.0 [M+HNa] (calcd for C H BrF NNaO S:
1
5
11
2
2
4
08.96).
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3-bromo-5-chloro-7-fluoro-1-tosyl-1H-indole (7b). To a solution of 5-chloro-7-fluoro-
-tosyl-1H-indole (3.65 g, 11.27 mmol) in DMF (23 mL) was added N-bromosuccinimide (2.41
1
g, 13.53 mmol) at room temperature and stirred at 50°C for 1h, then at ambient temperature for
16h. The mixture was poured into ice water and stirred for 30 min. The precipitate was isolated
1
by filtration to afford the titled compound as a tan solid (4.20 g, 93%). H NMR (400 MHz,
DMSO-d ) δ 8.28 (s, 1H), 7.86 (d, J=7.70 Hz, 2H), 7.41-7.50 (m, 3H), 7.36 (d, J=1.98 Hz, 1H),
6
-
2.35 (s, 3H). MS (ESI) m/z: 399.9 [M-H] (calcd for C H BrClFNO S: 399.92).
1
5
9
2
3
-bromo-5-methyl-7-fluoro-1-tosyl-1H-indole (7c). N-Bromosuccinimide (2.27 g, 12.75
mmol) was added to a solution of 7-fluoro-5-methyl-1-tosyl-1H-indole (4.07 g, 12.75 mmol) in
CH Cl (66 mL) at room temperature. The reaction mixture was stirred at room temperature for 18
2
2
hours. The mixture was treated with NaHCO (sat., aq.) and the mixture was stirred for 5 min. The
3
organic layer separated, dried over MgSO , the solids were removed by filtration and the solvent
4
was evaporated to dryness. The crude was purified by silica column chromatography using a
heptane to EtOAc gradient. Pure fractions were collected, and the solvent was evaporated. The
residue was crystallized from EtOH, isolated by filtration, and dried under vacuum to give 3-
1
bromo-7-fluoro-5-methyl-1-tosyl-1H-indole as beige powder (3.30 g, 68%). H NMR (400 MHz,
DMSO-d ) δ ppm 8.13 (s, 1 H) 7.83 (d, J=8.1 Hz, 2 H) 7.44 (d, J=8.6 Hz, 2 H) 7.12 (s, 1 H) 7.10
6
+
(
d, J=13.6 Hz, 1 H) 2.37 (s, 3 H) 2.35 (s, 3 H). MS (ESI) m/z: 399.3 [M+NH ] (calcd for
4
C H BrFN O S: 399.02).
1
6
17
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3
-bromo-7-fluoro-1-tosyl-1H-indole-5-carbonitrile (7d). To a solution of 7-fluoro-1-
tosyl-1H-indole-5-carbonitrile (1.70 g, 5.48 mmol) in CH Cl (15 mL) was added Br (0.33 mL,
2
2
2
6
.50 mmol) at 0°C. The mixture was stirred at 0°C for 30 minutes and then at room temperature
0
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for one additional hour. The reaction mixture was treated with a saturated solution of aqueous
NaHCO . The organic layer was separated and washed with aqueous Na S O , brine and water.
3
2
2
3
The organic layer was dried over MgSO , and concentrated. The crude was purified by silica
4
column chromatography using a heptane to EtOAc gradient. The desired fractions were collected
and evaporated to dryness to afford 3-bromo-7-fluoro-1-tosyl-1H-indole-5-carbonitrile (1.6 g,
1
7
6%). H NMR (400 MHz, DMSO-d ) δ 8.43 (s, 1H), 7.95 (d, J=1.32 Hz, 1H), 7.92 (d, J=7.70
6
Hz, 2H), 7.86 (dd, J=1.21, 11.99 Hz, 1H), 7.49 (d, J=8.14 Hz, 2H), 2.38 (s, 3H). MS (ESI) m/z:
-
391.1 [M-H] (calcd for C H BrFN O S: 390.96).
1
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,7-difluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole (8a).
A mixture of 3-bromo-5,7-difluoro-1-tosyl-indole (2 g, 5.18 mmol), bis-pinacolatodiboron (1.45
g, 5.70 mmol), Pd(dppf)Cl (76 mg, 0.10 mmol), 1,1’-bis(diphenylphosphino)ferrocene (55 mg,
2
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.10 mmol) and KOAc (1.01 g, 10.36 mmol) in dioxane (20 mL) was heated to 90°C for 18h under
nitrogen. The reaction cooled to room temperature, the solids were removed by filtration, and the
solvent of the filtrate was removed under reduced pressure. The crude was purified by silica
column chromatography using a heptane to CH Cl gradient. The desired fractions were collected
2
2
1
and evaporated to dryness to afford the titled compound as an off-white solid (1.99 g, 89%). H
NMR (400 MHz, DMSO-d ) δ 8.12 (s, 1H), 7.89 (d, J=7.70 Hz, 2H), 7.44 (d, J=7.92 Hz, 2H), 7.36
6
(
dd, J=2.31, 8.47 Hz, 1H), 7.21 (ddd, J=2.42, 9.68, 12.10 Hz, 1H), 2.36 (s, 3H), 1.32 (s, 12H). MS
+
(ESI) m/z: 434.0 [M+H] (calcd for C H BF NO S: 434.14).
21
23
2
2
5
-chloro-7-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole
(8b). The titled compound was prepared in a method analogous to that described for the
1
preparation of 8a with the exception that the reaction was heated to 90°C for 16 hours (45%). H
NMR (400 MHz, DMSO-d ) δ 8.14 (s, 1H), 7.89-7.94 (m, 2H), 7.64 (d, J=1.76 Hz, 1H), 7.45-7.48
6
(m, 2H), 7.35 (dd, J=1.87, 11.77 Hz, 1H), 2.38 (s, 3H), 1.35 (s, 12H). MS (ESI) m/z 450.0 [M+H]+
(
calcd for C H ClFNO S: 450.11).
21 23 4
7-fluoro-5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole
8c). A mixture of 3-bromo-7-fluoro-5-methyl-1-tosyl-1H-indole (0.7 g, 1.83 mmol),
(
bis(pinacolato)diboron (0.7 g, 2.75 mmol), Pd(dppf)Cl (0.075 g, 0.092 mmol) and potassium
2
acetate (0.54 g, 5.49 mmol) in DME (16 mL) was purged with N flow for 5 min and then was
2
stirred and heated at 100 °C for 18 h. EtOAc (40 mL) was added, the reaction mixture was filtered
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through packed celite, the filtrate was washed with brine, dried (MgSO ), the solids were removed
4
by filtration, and the filtrate was evaporated to dryness. The crude was purified by silica column
chromatography using a heptane to EtOAc gradient affording the titled compound (1.27 g, 96%).
0
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1
H NMR (400 MHz, DMSO-d ) δ ppm 7.99 (s, 1 H) 7.85 (d, J=8.1 Hz, 2 H) 7.37 - 7.49 (m, 3 H)
6
+
6
.92 - 7.03 (m, 1 H) 2.36 (s, 3 H) 2.34 (s, 3 H) 1.33 (s, 12 H). MS (ESI) m/z: 430.3 [M+H] (calcd
for C H BFNO S: 430.17).
2
2
26
4
7
-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole-5-
carbonitrile (8d). 3-bromo-7-fluoro-1-tosyl-1H-indole-5-carbonitrile (1.62 g, 4.11 mmol), bis-
pinacolatodiboron (3.13 g, 12.32 mmol), Pd(dppf)Cl (300 mg, 0.41 mmol) and KOAc (1.81 g,
2
1
8.48 mmol) in dioxane (15 mL) was heated to 80°C for 16h under nitrogen. The reaction was
cooled to room temperature, the solids removed by filtration, and the solvent of the filtrate was
removed under reduced pressure. The crude was purified by silica column chromatography using
a heptane to EtOAc gradient. The desired fractions were collected and evaporated to dryness to
afford 7-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole-5-carbonitrile
1
(
1.1 g, 60%). H NMR (400 MHz, DMSO-d ) δ 8.25 (s, 1H), 8.02 (d, J=1.32 Hz, 1H), 7.95 (d,
6
J=7.26 Hz, 2H), 7.76 (dd, J=1.21, 11.99 Hz, 1H), 7.48 (d, J=8.14 Hz, 2H), 2.38 (s, 3H), 1.35 (s,
+
1
2H). MS (ESI) m/z: 441.2 [M+H] (calcd for C H BFN O S: 441.15).
2
2
23
2
4
Methyl
(2S,3S)-3-((2-(5,7-difluoro-1-tosyl-1H-indol-3-yl)-5-fluoropyrimidin-4-
yl)amino)bicyclo[2.2.2]octane-2-carboxylate (10a). A mixture of 5,7-difluoro-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole (0.4 g, 0.92 mmol), (2S,3S)-3-((2-chloro-
5
-fluoropyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (316 mg, 1 mmol) and
potassium phosphate (588 mg, 2.8 mmol) was stirred in water (1.2 mL) and 1,4-dioxane (6 mL) at
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room temperature under nitrogen. Then PdCl (dtbpf) (133 mg, 0.2 mmol) was added and stirred
2
under nitrogen for 10 minutes. The vessel was closed, and the reaction was heated in the
microwave for 10 minutes at 130°C. The mixture was diluted with EtOAc and methyl-THF. Dried
1
1
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over MgSO , filtered over celite and the solvent of the filtrate was removed under reduced
4
pressure. The crude was purified by silica column chromatography using a heptane to EtOAc
gradient. The corresponding fractions were evaporated and dried in vacuo to afford methyl
(2S,3S)-3-((2-(5,7-difluoro-1-tosyl-1H-indol-3-yl)-5-fluoropyrimidin-4-
1
yl)amino)bicyclo[2.2.2]octane-2-carboxylate (0.539 g, 68%). H NMR (400 MHz, DMSO-d ) δ
6
8
7
.53 (s, 1H), 8.28 (d, J=3.74 Hz, 1H), 8.23 (dd, J=2.42, 9.02 Hz, 1H), 7.91 (d, J=7.70 Hz, 2H),
.84 (br d, J=6.82 Hz, 1H), 7.48 (d, J=8.14 Hz, 2H), 7.28 (ddd, J=2.31, 9.41, 12.05 Hz, 1H), 4.73
(br t, J=6.93 Hz, 1H), 3.65 (s, 3H), 3.00 (br d, J=6.82 Hz, 1H), 2.38 (s, 3H), 1.98 (br d, J=16.73
+
Hz, 2H), 1.72-1.88 (m, 3H), 1.36-1.68 (m, 5H). MS (ESI) m/z: 585.4 [M+H] (calcd for
C H F N O S: 585.18).
2
9
28
3
4
4
Methyl
(2S,3S)-3-((2-(5-chloro-7-fluoro-1H-indol-3-yl)-5-fluoropyrimidin-4-
yl)amino)bicyclo-[2.2.2]octane-2-carboxylate (10b). The titled compound was prepared in a
method analogous to that described for the preparation of 10a with the exception that in step one,
the reaction was heated at 100°C for 15 min under microwave irradiation and 0.8 equivalent of
(2S,3S)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate was used
1
(
39 %). H NMR (400 MHz, DMSO-d ) δ 8.50 (d, J=1.76 Hz, 1H), 8.48 (s, 1H), 8.27-8.29 (m,
6
1H), 7.89 (d, J=8.14 Hz, 2H), 7.84 (br d, J=6.60 Hz, 1H), 7.47 (d, J=8.14 Hz, 2H), 7.40-7.44 (m,
1
H), 4.71 (br t, J=7.15 Hz, 1H), 3.61 (s, 3H), 2.93-2.98 (m, 1H), 2.38 (s, 3H), 1.98 (br s, 1H), 1.87-
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+
1.93 (m, 1H), 1.70-1.87 (m, 3H), 1.38-1.67 (m, 5H). MS (ESI) m/z: 601.1 [M+H] (calcd for
C H ClF N O S: 601.18).
29 28
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Methyl
yl)amino)bicyclo-[2.2.2]octane-2-carboxylate (10c). A solution of 7-fluoro-5-methyl-3-
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole (1.09 g, 1.26 mmol), methyl
(2S,3S)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (0.33 g,
(2S,3S)-3-((5-fluoro-2-(7-fluoro-5-methyl-1H-indol-3-yl)pyrimidin-4-
(
1
0
5
.05 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).CH Cl (0.086 g,
2 2
.11 mmol) in K CO (2M, aq., 2.11 mL, 4.21 mmol) and DME (14 mL) was purged with N for
2
3
2
min and then was heated at 100°C using a microwave for 30 min. The mixture was poured into
water and CH Cl , the organic layer was separated and evaporated to dryness. The crude was
2
2
purified by silica column chromatography using a heptane to EtOAc gradient. Pure fractions were
collected, and the solvent was evaporated to give the titled compound as a white solid (0.27 g,
1
44%). H NMR (400 MHz, DMSO-d ) δ 8.39 (s, 1H), 8.26 (s, 1H), 8.26 (d, J=3.65 Hz, 1H), 7.85
6
(
d, J=7.92 Hz, 2H), 7.79 (br d, J=7.26 Hz, 1H), 7.44 (d, J=8.14 Hz, 2H), 7.04 (d, J=12.98 Hz, 1H),
4
.77 (br t, J=6.93 Hz, 1H), 3.61 (s, 3H), 2.95 (br d, J=7.04 Hz, 1H), 2.40 (s, 3H), 2.36 (s, 3H), 1.97
(br s, 1H), 1.89 (br s, 1H), 1.69-1.83 (m, 3H), 1.32-1.67 (m, 5H). MS (ESI) m/z: 581.0
+
[
M+H] (calcd for C H F N O S: 581.20).
3
0
31
2
4
4
Methyl
(2S,3S)-3-((2-(5-cyano-7-fluoro-1H-indol-3-yl)-5-fluoropyrimidin-4-
yl)amino) bicyclo[2.2.2]octane-2-carboxylate (10d). A solution of 7-fluoro-5-cyano-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-indole (0.20 g, 0.45 mmol), methyl (2S,3S)-3-
((2-chloro-5-fluoropyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (0.14 g, 0.45 mmol)
and Pd(dppf)Cl (0.030 g, 0.05 mmol) and K PO (0.29 g, 1.36 mmol) in 1,4-dioxane (4 mL) and
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H O (0.4 mL) was purged with N for 5 minutes and then heated to 100°C in the microwave for
2
2
30 minutes. The mixture was poured into water and CH Cl , the organic layer was separated and
2
2
evaporated to dryness. The crude was purified via silica gel chromatography using a heptane to
EtOAc gradient. Pure fractions were collected, and the solvent was removed under reduced
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
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7
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3
4
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8
9
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1
2
3
4
5
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9
0
1
pressure to afford the titled compound as a white solid (0.11 g, 41%). H NMR (400 MHz, DMSO-
d6) δ 8.87 (d, J=1.54 Hz, 1H), 8.58 (s, 1H), 8.30 (d, J=3.96 Hz, 1H), 7.94 (d, J=7.48 Hz, 2H), 7.89
(
br d, J=7.26 Hz, 1H), 7.82 (dd, J=1.21, 11.99 Hz, 1H), 7.48 (d, J=8.14 Hz, 2H), 4.74 (br t, J=7.48
Hz, 1H), 3.61 (s, 3H), 2.95 (br d, J=7.26 Hz, 1H), 2.39 (s, 3H), 1.96-2.03 (m, 1H), 1.88-1.96 (m,
H), 1.69-1.86 (m, 3H), 1.37-1.69 (m, 5H). MS (ESI) m/z: 592.0 [M+H]+ (calcd for
1
C H F N O S: 592.19).
3
0
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(
2S,3S)-3-((2-(5,7-difluoro-1H-indol-3-yl)-5-fluoropyrimidin-4-yl)amino)bicyclo
[2.2.2]octane-2-carboxylic acid (11a). Methyl (2S,3S)-3-((2-(5,7-difluoro-1-tosyl-1H-indol-3-
yl)-5-fluoropyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (4 g, 6.84 mmol) was
stirred in 1,4-dioxane (110 mL) at 60°C, while a solution of LiOH (1.64 g, 68.42 mmol) in water
(20 mL) was added. The mixture was brought to reflux for ten minutes and stirred overnight at
room temperature. The solvent was removed under reduced pressure and the crude was
reconstituted in water (150 mL), stirred and neutralized with HCl (1N aq., 70 mL). The precipitate
was isolated by filtration and a purification was performed via preparatory HPLC (Uptisphere C18
ODB – 10 µm, 200 g, 5 cm, mobile phase: 0.25% NH HCO solution in water, CH CN) to the
0
1
2
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0
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afford the titled compound (2.01 g, 71%). H NMR (360 MHz, DMSO-d ) δ 12.19 (br s, 1H), 8.15
6
(
d, J=4.02 Hz, 1H), 8.06-8.12 (m, 2H), 7.56 (br d, J=6.59 Hz, 1H), 7.02-7.09 (m, 1H), 4.69 (br t,
J=6.77 Hz, 1H), 2.86 (br d, J=6.95 Hz, 1H), 1.80-2.03 (m, 3H), 1.69-1.79 (m, 2H), 1.30-1.68 (m,
13
6
1
H). C NMR (101 MHz, DMSO-d ) δ 176.0, 158.6, 151.7, 145.2, 142.7, 139.0, 138.8, 131.0,
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03.1, 102.9, 97.3, 97.0, 50.8, 48.1, 28.8, 28.8, 28.7, 25.7, 24.1, 21.6, 19.5. ESI-HRMS (TOF) m/z:
+
20
417.1536 [M+H] (Calcd. for C H F N O : 417.1460). [α] 38.5 (c 0.8, DMF).
2
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20
3
4
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D
(
2S,3S)-3-((2-(5-chloro-7-fluoro-1H-indol-3-yl)-5-fluoropyrimidin-4-
yl)amino)bicyclo-[2.2.2]octane-2-carboxylic acid (11b). The titled compound was prepared in a
1
method analogous to that described for the preparation of 11a (79%). H NMR (400 MHz, DMSO-
d6) δ 8.38 (d, J=1.76 Hz, 1H), 8.11 (d, J=3.96 Hz, 1H), 8.09 (s, 1H), 7.39 (br d, J=6.82 Hz, 1H),
7
.14 (dd, J=1.76, 10.78 Hz, 1H), 4.72 (br t, J=6.71 Hz, 1H), 2.67 (br d, J=6.60 Hz, 1H), 1.88-2.06
1
3
(
m, 3H), 1.28-1.78 (m, 7H). C NMR (151 MHz, DMSO-d ) δ 177.1, 158.4, 151.8, 149.1, 144.0,
6
138.5, 131.0, 129.8, 124.4, 123.9, 117.7, 116.4, 107.9, 51.7, 49.4, 28.9, 28.8, 26.1, 24.5, 21.9, 19.8.
+
ESI-HRMS (TOF) m/z: 433.1245 [M+H] (Calcd. for C H ClF N O : 433.1165).
2
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19
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ACS Paragon Plus Environment

Page 29 of 51
Journal of Medicinal Chemistry
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(
2S,3S)-3-((5-fluoro-2-(7-fluoro-5-methyl-1H-indol-3-yl)pyrimidin-4-
yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid (11c). A solution of methyl (2S,3S)-3-((5-
fluoro-2-(7-fluoro-5-methyl-1-tosyl-1H-indol-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-
carboxylate (1.25 g, 2.15 mmol) and KOH powder (1.21 g, 21.53 mmol) in EtOH (13 mL) was
stirred and heated at 80°C for 40 min. The solution was cooled to room temperature and the solvent
was evaporated. The residue was taken up in water (40 mL) and neutralized with HCl (aq., 1N, 21
mL). The mixture was stirred for 10 minutes, and then the precipitate was isolated by filtration and
dried under vacuum at 50°C affording a beige solid. The crude was purified by silica column
chromatography using a CH Cl to 7% CH OH in CH Cl gradient. Pure fractions were collected,
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the solvent was removed under reduced pressure. The compound was freeze-dried with
1
acetonitrile/water: 2/8 to afford the titled compound as a white powder (62 mg, 7%). H NMR (400
MHz, DMSO-d ) δ 11.86 (d, J=2.00 Hz, 1H), 8.15 (s, 1H), 8.12 (d, J=3.96 Hz, 1H), 7.96 (d, J=2.64
6
Hz, 1H), 7.47 (d, J=7.00 Hz, 1H), 6.83 (d, J=12.10 Hz, 1H), 4.77 (t, J=6.80 Hz, 1H), 2.84 (d,
1
3
J=6.80 Hz, 1H), 2.42 (s, 3H), 1.88-2.04 (m, 2H), 1.27-1.87 (m, 8H). C NMR (101 MHz, DMSO-
d6) δ 176.1, 159.1, 151.7, 150.4, 148.0, 145.1, 142.6, 138.9, 130.2, 129.6, 123.4, 117.9, 108.5,
0.8, 49.1, 48.3, 28.8, 25.8, 24.5, 21.7, 21.6, 19.4. ESI-HRMS (TOF) m/z: 413.1789 [M+H]+
5
(
Calcd. for C H F N O : 413.1711).
22 23 2 4 2
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