Synthesis and biological evaluation of substituted 3-anilino-quinolin-2(1H) -ones as PDK1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.04.037

PDK1 is an important regulator of the PI3K/Akt pathway, which has been found frequently activated in a large number of human cancers. Herein we described the preparation of novel substituted 3-anilino-quinolin-2(1H)-ones as PDK1 inhibitors. The synthesis

Full text of DOI:10.1016/j.bmc.2014.04.037

Accepted Manuscript
Synthesis and biological evaluation of substituted 3-anilino-quinolin-2(1H)-
ones as PDK1 inhibitors
Nathan J. O’Brien, Martin Brzozowski, David J.D. Wilson, Leslie W. Deady,
Belinda M. Abbott
PII:
S0968-0896(14)00302-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.04.037
BMC 11532
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 February 2014
17 April 2014
18 April 2014
Please cite this article as: O’Brien, N.J., Brzozowski, M., Wilson, D.J.D., Deady, L.W., Abbott, B.M., Synthesis
and biological evaluation of substituted 3-anilino-quinolin-2(1H)-ones as PDK1 inhibitors, Bioorganic & Medicinal
Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.04.037
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Synthesis and biological evaluation of
substituted 3-anilino-quinolin-2(1H)-ones as
PDK1 inhibitors
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Nathan J. O’Brien, Martin Brzozowski, David J.D. Wilson, Leslie W. Deady and Belinda M. Abbott
Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Vic 3086, Australia

1
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis and biological evaluation of substituted 3-anilino-quinolin-2(1H)-ones as
PDK1 inhibitors
Nathan J. O’Brien, Martin Brzozowski, David J.D. Wilson, Leslie W. Deady and Belinda M. Abbott
aDepartment of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Vic 3086, Australia
ARTICLE INFO
ABSTRACT
PDK1 is an important regulator of the PI3K/Akt pathway, which has been found frequently activated
in a large number of human cancers. Herein we described the preparation of novel substituted 3-
anilino-quinolin-2(1H)-ones as PDK1 inhibitors. The synthesis is based around a Buchwald-Hartwig
cross-coupling of various 3-bromo-6-substituted-quinolin-2(1H)-ones with three different
functionalised anilines. The modular nature of the designed synthesis allowed access to a series of
novel inhibitors through derivatisation of a late-stage intermediate. All compounds were screened
against isolated PDK1 enzyme, with modest inhibition observed.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
3-Phosphoinositide-dependent kinase 1 (PDK1)
3-Anilino-quinolin-2(1H)-ones
Inhibitor
2009 Elsevier Ltd. All rights reserved.
Buchwald-Hartwig cross-coupling
Cross-coupling
1. Introduction
A key area of the ATP binding site is the hinge region, where
an inhibitor can lock in through hydrogen bonding interaction
3-Phosphoinositide-dependent kinase 1 (PDK1) is the master
regulator of the AGC signal transduction and is responsible for
the activity of as many as 23 different agonist-stimulated AGC
kinases, including Akt,1,2 PKC isoforms,3 P70 S6K,4 p90 SGK5
and RSK6 by phosphorylation at its own activation loop.7,8 These
AGC kinases are members of the PI3K/Akt signalling pathway
and are constitutively activated in a large number of human
cancers in response to abnormally high levels of the secondary
messenger, phosphatidylinositol 3,4,5-triphosphate (PIP3).9,10
PDK1 plays a vital role in the positive regulation of the important
cellular effects triggered by the PI3K/Akt signalling pathway and
with the Ser-89, Tyr-90 and Ala-91 residues. Binding potency
and selectivity can then be enhanced by additional interactions
with other areas in or near the active site. We recently reported
the synthesis of novel compounds containing 4-(1H-indol-6-yl)-
1H-indazoles18 as potential PDK1 inhibitors. These compounds
were designed to have a heterocyclic core bound to the hinge
region, with additional functionality to target various binding site
pockets. In this contemporary study, again guided by molecular
modelling of ligand interactions, a 3-anilino-quinolin-2(1H)-one
core 1 was identified to also possess an appropriate geometry and
electronic properties for the binding site (Figure 1).
subsequent
PIP3
production.8,11
Activation
of
phosphatidylinositol 3-kinase (PI3K) stimulates the production of
PIP3, which interacts with several downstream effector proteins
to control the activity and subcellular localisation of a diverse
range of signal transducers that are fundamental to cell growth,
metabolism, proliferation and survival.8,12,13
Disruption or dysregulation of this pathway, through gene
mutation, leads to the promotion of tumour proliferation, reduced
apoptosis and angiogenesis.14,15 Recent experimental evidence
has shown that specific inhibitors of this pathway are able to
promote apoptosis in cells and reduce tumour growth.16 As PDK1
is the critical regulator within the PI3K/Akt pathway, targeted
inhibition of this downstream enzyme represents a promising
biological target for the development of anti-cancer drugs.14,17
Fig. 1. Schematic representation of the 3-anilino-quinolin-2(1H)-one 1 core
bound to the hinge region (Ser-89, Tyr-90, Ala-91) of PDK1 binding site.
———
 Corresponding author. Tel.: +61 3 94792520; fax: +61 3 94791266; e-mail: b.abbott@latrobe.edu.au

2
The nitrogens and the carbonyl of the quinolin-2(1H)-one
Herein, the synthetic route to this novel series of
heterocyclic compounds is reported, along with results for
inhibition of activity towards isolated PDK1 enzyme.
backbone and 3-position aniline provide important interactions
with the hinge region, locking the inhibitor into the binding
site. As suggested by molecular modelling, additional
functionality was added to the inhibitor core: access to the
solvent exposed entrance regions (E0 and E1) and the ribose
pocket (R) was targeted by adding functionality to the phenyl
ring of the 3-position aniline (R2 and R3). Specific targeting of
the catalytic residues (Cat.) of the phosphate pocket (P) and
hydrophobic region (BP-I) was attempted by appending an
appropriate functional group to the 6-position of the quinolin-
2(1H)-one scaffold (R1).
2. Results and Discussion
The synthetic strategy for the 3-anilino-quinolin-2(1H)-one
derivatives 1a-h was designed on the independent synthesis of
two key fragments and then linking them together by a
Buchwald-Hartwig cross-coupling reaction to form a new C-N
bond (Scheme 1 and Table 2). The functionalised quinolin-
2(1H)-one intermediate 4 was an appropriate heterocyclic core
for the planned cross-coupling reaction with various
substituted anilines 5. Incorporation of a protecting group (PG)
for this fragment was deemed to be a requirement for quinolin-
2(1H)-ones when undertaking palladium catalysed cross-
Compound 2 and 3 (Figure 2), potent PDK1 inhibitors
developed by Berlex Biosciences,19,20 were identified from
recent literature, both with X-ray crystal structures available in
complex with PDK1 (PDB codes: 1Z5M and 2PE2).
Compound 2 contains a pyrrolidine urea moiety that binds to
the ribose pocket of PDK1, whilst compound 3 contains the
N-(2-(piperidin-1-yl)ethyl)acetamide chain that binds to the E0
region and provides further interactions with the E1 region.
coupling reactions.21,22 Following literature precedence,23-25
a
p-methoxybenzyl (PMB) group was chosen due to its ease of
formation and cleavage from amides of quinolinones.
Fig. 2. Known PDK1 inhibitors developed by Berlex Biosciences.
A methylene linker was suggested by our modelling to give
the appropriate chain length to reach the E0 region from the 3-
aniline ring. The pyrrolidine urea was incorporated onto the
phenyl ring of the 3-position aniline (R2 position) of target
compound 1a whilst both side chains were included (R2 and R3
positions) for target compound 1b (Figure 3a and 3b,
respectively, and Table 2). Compound 3 also contains a
primary urea group, which provides key hydrogen bond
interactions with the catalytic residues of the phosphate pocket,
and this was incorporated at the 6-position (R1 position) of the
quinolinone scaffold for target compounds 1a and b. For
comparison, the primary urea at the R1 position of the scaffold
without any substitutions at either the R2 and R3 positions
would also be undertaken. Replacement of the primary urea
with a diverse group of R1 substituents would enable the effect
of such changes on biological activity to be further probed.
Scheme 1. Retrosynthetic strategy towards the novel substituted 3-anilino-
quinolin-2(1H)-ones.
2-Hydroxyquinoline 6 was treated under standard nitration
conditions to give 6-nitroquinolinone 7 in 95% yield (Scheme
2). Acid catalysed bromination, through in situ generation of
hypobromous acid, furnished the desired 3-bromo-6-
nitroquinolinone 8 in a yield of 87%.26 Synthesis of compound
8 using these conditions had been previously reported by Lee
et al.27, but no experimental or characterisation detail was
reported. Benzylation of 8 with p-methoxybenzyl chloride and
potassium carbonate gave the N-PMB protected quinolinone
9.28,29
a)
b)
Fig. 3. Molecular model of target 3-anilino-quinolin-2(1H)-ones 1a (a) and 1b (b) bound to PDK1 illustrating access to the hinge region (Hinge), catalytic
residues (Cat.), ribose pocket (R), and entrance regions (E0 and E1) from the designed scaffold. The vector in a) indicates extension of the scaffold at C3 by
addition of the R3 side chain.

3
Metal catalysed reduction of compound 9 to the corresponding
amine 10 was achieved in 80% yield with 10 equivalents of
zinc powder in a 1:3 mixture of acetic acid and methanol at
room temperature.30
removed by flash chromatography on a small scale (approx.
200 mg) due to the insolubility of the mixture. Installation of
an ethoxy group was achieved by using a copper iodide/1,10-
phenanthroline catalysed alkoxylation method reported by
Buchwald et al.37 to give a 1:1:1 mixture of the desired 6-
ethoxy analogue 11e, along with the 3-ethoxy-6-iodo 13a and
3,6-diethoxy 13b side-products which were all isolated by
flash chromatography and characterised. The lack of
regioselectivity between 3- and 6-positions was a noteworthy
difference from the previous cyanation and Suzuki reactions.
Attempts to install an alkoxy group by modified Suzuki cross-
Amine 10 provided access to the 6-ureido compound 11a in
80% yield, by treatment with isocyanuric acid produced in situ.
1
Acetanilide 11b was observed in the H-NMR spectrum of the
crude material, but this minor side-product was successfully
removed by recrystallisation. Generation of a reactive mixed
anhydride (H2NCOOCOCH3) from the isocyanuric acid and
acetic acid was a plausible explanation for the unwanted
acetanilide formation. Formation of an acetanilide side-product
was also observed by Silvestri et al.31 when forming ureas with
isocyanuric acid. Following conditions previously utilised by
our laboratory,32 amine 10 was also able to be acetylated to
give 11b in a quantitative yield.
coupling
conditions38-41
with
potassium
methoxymethyltrifluoroborate were all unsuccessful.
The 3-anilino-quinolin-2(1H)-ones 1a-g were synthesised
by Buchwald-Hartwig cross-coupling of bromo compounds
11a-11e with anilines 14 and 15 (Figure 3), that were
synthesised by our laboratory,42 as well as aniline, followed by
an acid catalysed debenzylation.
Fig. 3. Custom anilines 14 and 15.
Buchwald-Hartwig cross-coupling conditions43 yielded the
N-PMB protected quinolinones 16a-16g in yields ranging from
44-92% (Table 2). These conditions provided a highly efficient
and reliable alternative for synthesising 3-anilino-quinolin-
2(1H)-ones over conventional methods44-52 which require
multi-step procedures that suffer from low yields and limited
substrate scope. Due to the insolubility of urea 11a, the yields
for intermediates 16a-16c were initially lower than desired but
were improved when the amount of reaction solvent was
doubled. Tetrazole compound 16h was synthesised in 98%
yield by a [3 + 2] cycloaddition53 of nitrile intermediate 16e
Scheme 2. Reagents and Conditions: (a) cH2SO4/cHNO3, 0 ˚C, 30 min,
95%; (b) NaBrO3 (1.5 eq.), 48% aq. HBr, H2O, 100 ˚C, 16 h, 87%; c)
PMB-Cl (1.5 eq.), K2CO3 (1.5 eq), DMF, rt, 5 h; then rt  50 ˚C, 16 h,
90%; (d) Zn (10 eq.), 1:3 AcOH/MeOH, rt, 30 min, 80%; (e) NaOCN (3
eq.), 4:1 AcOH/H2O, rt, 1 h, 80%; (f) AcCl, Et3N, THF, 0 ˚C, 30 min; then
0 ˚C  rt, 30 min, 98%.
Installation of an iodine moiety at the 6-position provided a
handle for the introduction of a more diverse range of
functional groups via cross-coupling chemistry. Insight into the
relative reactivity of the 6-iodo versus the 3-bromo position
was also obtained. While 3-bromo-6-iodoquinolin-2(1H)-one
has been prepared, starting with 4-iodoaniline,33 amine 10
provided access to this scaffold without starting a new
synthetic pathway. Diazotization of amine 10 with sodium
nitrite and then treatment with potassium iodide provided the
iodo compound 12 in a 90% yield (Scheme 3).34
with
trimethylsilyl
azide
in
the
presence
of
tetrabutylammonium fluoride.
Table 2. Synthesis of N-PMB protected quinolinones 16a-g by Buchwald-
Hartwig cross-coupling and tetrazole compound 16h by [3
+ 2]
cycloaddition with TMS-N3.
Scheme 3. Reagents and Conditions: (a) NaNO2 (2 eq.), cHCl, -5 ˚C, 1 h;
KI (25 eq.), H2O, -5 ˚C  rt, 4 h, 91%; (b) Zn(CN)2 (0.65 eq.), Pd(PPh3)4
(7.5 mol%), DMF, 80 ˚C, 4 h, 82%; (c) 3-MeOC6H4B(OH)2 (1.2 eq.),
Pd(PPh3)4 (5 mol%), 4:1 1,4-dioxane/H2O, 90 ˚C, 16 h, 52%; (d) CuI (10
mol%), 1,10-phenanthroline (20 mol%), Cs2CO3 (2 eq.), EtOH, reflux, 16
h, 35%.
Bromo cmpd
Aniline
Product
Yield %
11a
11a
11a
11b
11c
11d
11e
14
15
PhNH2
14
14
14
16a
16b
16c
16d
16e
16f
51
58
44
81
92
73
78
Nitrile installation using palladium catalysed cyanation
conditions35 with iodo 12 and zinc cyanide gave nitrile 11c
with high selectivity to the 6-position. A small amount (circa
5-10%) of the 3,6-disubstituted side-product was observed by
1H-NMR, but this was easily removed by flash
chromatography. Modified Suzuki cross-coupling36 of 3-
methoxyphenylboronic acid to iodo 12 afforded the 6-(3-
methoxyphenyl) analogue 11d, with a small amount (circa 10-
15%) of the 3,6-disubstituted side-product which could only be
14
16g
Reagents and Conditions: (a) aniline (1.2 eq.), K2CO3 (2.2 eq.), Pd2(dba)3
(5 mol%), XPhos (10 mol%), t-BuOH, reflux, 16 h; (b) TMS-N3 (10 eq.),
TBAF (1.2 eq.), 80 ˚C, 48 h, 98%.

4
Table 3. Synthesis of 3-anilino-quinolin-2(1H)-ones 1a-h by acid catalysed debenzylation, and their percentage inhibitory activity against PDK1.
% Inhibition at 10 Ma
405
Compound
R1
R2
R3
H
Yield %
65
1a
1b
1c
1d
1e
1f
26b
47
62
29
55
60
406
368
206
307
298
238
H
H
H
H
H
H
1g
1h
H
34
147
Reagents and Conditions: (a) TfOH (3.5 eq.), TFA, rt, 3 h. aThe mean  SD of at least 3 independent experiments (radiometric assay). bRequired purification by
RP-HPLC.
Treatment of N-PMB protected quinolinones 16a-g with
triflic acid in trifluoroacetic acid54 furnished the desired novel
3-anilino-quinolin-2(1H)-ones 1a-g in moderate yields (Table
3). Trifluoroacetic acid alone, commonly used for this type of
slightly decreased the activity of 1c. However, changes to the
6-position of the quinolin-2(1H)-one scaffold had a significant
impact on the activity of this class of compounds and reduced
the inhibition of compounds 1d-h towards PDK1. Direct
derivatisation to an acetanilide 1d decreased activity to 20±6%,
a 2-fold decrease in comparison to the designed target
compounds 1a and 1b. This shows the importance of the
hydrogen bond donors from the NH2 moiety of the urea to
activity. Addition of a 3-methoxyphenyl moiety 1f decreased
the inhibition to 29±8%, whilst an ethoxy group 1g gave a
similar result of 23±8%. Installation of a nitrile moiety 1e also
showed a reduction of inhibition (30±7%), whilst further
transformation to a tetrazole 1h gave the poorest result
(14±7%). Even though the tetrazole is a potential hydrogen
bond donor, its high insolubility profile was a plausible
explanation behind its lack of activity.
debenzylations,55-61 failed though, for example,
9 was
debenzylated successfully. Thus, the new 3-aniline linker was
the problem. It was reasoned that trifluoroacetic acid was
monoprotonating compounds 16a-h at the exocyclic nitrogen
rather than the ring nitrogen, the latter being a necessary
condition for debenzylation. The stronger triflic acid seemingly
achieved this outcome through diprotonation. The use of the
Buchwald-Hartwig cross-coupling to install aniline
functionality at the 3-position of quinolin-2(1H)-ones has been
previously reported,21,22,62 but this is the first example to the
best of our knowledge where the protecting group has been
subsequently removed to furnish the N-unprotected product.
The highly insoluble nature of products 1e and 1h in organic
solvents made purification difficult and is the reason for the
lower yields obtained from these debenzylation reactions.
3. Conclusion
We have developed an efficient and reliable route for the
preparation of novel substituted 3-anilino-quinolin-2(1H)-ones.
Synthesis of a 6-aminoquinolinone intermediate gave access to
a range of 3-bromo-6-substituted-quinolin-2(1H)-ones, which
were cross-coupled at the 3-position with three different
anilines. Use of Buchwald-Hartwig cross-coupling over
conventional methods was highly effective. In addition,
utilisation of the PMB protecting group provided a versatile
system for the synthesis of various N-unprotected 3-anilino-
quinolin-2(1H)-ones. Selectivity for the 6-iodo over the 3-
Target compounds 1a-h were screened against isolated
PDK1 using a radiometric biological assay at a concentration
of 10 M (Table 3). Unfortunately, only modest inhibitory
activity was observed. The initial target compounds 1a and 1b
were the best inhibitors of the series, both possessing inhibition
of 405-6%, which illustrates the homologated N-(2-
(piperidin-1-yl)ethyl)acetamide side chain of 1b is not a
necessity for activity. Elimination of both the pyrrolidine urea
and N-(2-(piperidin-1-yl)ethyl)acetamide side chains only

5
bromo was shown for both palladium cross-coupling reactions,
with only small amounts of the 3,6-disubstituted side-products
observed. Unfortunately, the assessed compounds displayed
only modest inhibitory activity towards PDK1. The primary
urea moiety was found to be beneficial for binding interactions
with the catalytic residues over other 6-position substituents.
Further investigation into replacing the primary urea group at
the 6-position with various hydrogen bond donor groups could
enhance the binding potential of these compounds.
4.2.1 6-Nitroquinolin-2(1H)-one (7). To a stirring solution of
2-hydroxyquinoline 6 (3.50 g, 24.1 mmol) in conc. H2SO4 (15
mL) at -5 ºC, was added conc. HNO3 (500 µL) dropwise
ensuring the reaction temperature was < 20 ºC. The solution
was stirred at -5 °C for 30 min and then poured onto ice. The
precipitate that formed was filtered and washed with H2O to
give the 6-nitroquinolinone 7 (4.35 g, 95%) as a light green
solid; mp 279-281 ºC (from i-PrOH) (lit.65 mp 280 °C); H
(DMSO-d6): 12.26 (1H, s), 8.65 (1H, d, J 2.5 Hz), 8.29 (1H,
dd, J 9.0, 2.5 Hz), 8.09 (1H, d, J 9.5 Hz), 7.40 (1H, d, J 9.0
Hz), 6.65 (1H, d, J 9.5 Hz); C (DMSO-d6): 161.93, 143.2,
141.4, 140.1, 125.0, 124.2, 123.8, 118.5, 116.5. m/z (ESI):
191.0 (MH+).
4. Experimental Section
4.1 General Methods
Melting points were recorded on a Reichert “Thermopan”
microscope hot stage apparatus and are uncorrected. NMR
spectra were recorded on a Bruker AV-500 at 500.19 MHz for
1H nuclei and at 125.78 MHz for 13C nuclei at 300 K unless
otherwise stated. All of the chemical shifts were recorded as δ
values in parts per million (ppm) and coupling constants (J)
were recorded in hertz (Hz) to the nearest 0.5 Hz. The
4.2.2 3-Bromo-6-nitroquinolin-2(1H)-one (8). To a suspension
of the 6-nitroquinolinone 7 (4.35 g, 22.8 mmol), NaBrO3 (4.14
g, 27.4 mmol) and H2O (15 mL) was added 48% aq. HBr (80
mL) and the reaction mixture was heated at 100 °C for 4 h,
then cooled and poured onto ice. The precipitate that formed
was filtered and washed with H2O. The solid was recrystallised
from AcOH and EtOH to give the 3-bromo-6-nitroquinolinone
8 (5.35 g, 87%) as a white solid; mp >300 ºC; δH (DMSO-d6):
12.76 (1H, s), 8.72 (1H, d, J 4.5 Hz), 8.69 (1H, dd, J 4.5, 2.5
Hz), 8.35 (1H, ddd, J 9.0, 3.5, 2.5 Hz), 7.46 (1H, dd, J 9.0, 3.5
Hz); δC (DMSO-d6): 157.7, 142.3, 141.8, 141.4, 125.1, 123.6,
119.3, 118.8, 116.3; m/z (ESI): 269.0 (M[79Br]H+), 271.0
(M[81Br]H+); HRMS (ESI): M[79Br]H+, found 268.9555.
C9H6BrN2O3+ requires 268.9557.
1
following abbreviations were used when reporting H-NMR
data: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
app, apparent; and br, broad. Low-resolution electrospray
ionization (ESI) mass spectra were recorded on a Bruker
Daltronics Esquire 6000 ion trap mass spectrometer at 300 ˚C
with a scan rate of 5500 m/z/sec, a 40 eV cone voltage and in
positive mode, unless otherwise stated. Methanol or
acetonitrile with 0.1% formic acid was used as the mobile
phase. High-resolution electrospray were recorded on an
Agilent 1290 Infinity. All data were acquired and reference
mass corrected via a dual-spray electrospray ionization (ESI)
source, in positive mode, unless otherwise stated. Preparatory
RP-HPLC was performed on a Beckman system (125 Solvent
Module and 166 Detector) fitted with a Phenomenex® Jupiter
C18 300 Å column (250 mm x 10 mm, 10 µm) at a flow rate of
4.2.3 3-Bromo-1-(4-methoxybenzyl)-6-nitroquinolin-2(1H)-one
(9). To a solution of the 3-bromo-6-nitroquinolinone 8 (4.00 g,
14.8 mmol) in anhydrous DMF (40 mL) was added K2CO3
(3.08 g, 22.3 mmol) and p-methoxybenzyl chloride (3.02 mL,
22.3 mmol). The reaction was stirred at room temperature for 5
h and later heated to 50 °C overnight, then cooled and poured
onto ice. The precipitate that formed was filtered, washed with
H2O and recrystallised from EtOH to give the N-PMB
protected quinolinone 9 (5.20 g, 90%) as a fluffy white solid;
mp 208-209 °C; δH (DMSO-d6): 8.82 (1H, s), 8.74 (1H, d, J 2.5
Hz), 8.33 (1H, dd, J 9.5, 2.5 Hz), 7.65 (1H, d, J 9.5 Hz), 7.17
(2H, d, J 8.5 Hz), 6.87 (2H, d, J 8.5 Hz), 5.54 (2H, s), 3.69
(3H, s); δC (DMSO-d6): 158.5, 157.7, 142.3, 141.9, 141.2,
128.0, 127.4, 125.1, 124.3, 120.0, 118.4, 116.6, 114.1, 55.0,
46.5; m/z (ESI): 801.0 (M2[79,81Br]Na+), 411.0 (M[79Br]Na+),
413.0 (M[81Br]Na+); HRMS (ESI): M2[79,81Br]Na+, found
800.9980 (100%). C34H26Br2N4NaO8+ requires 800.9990.
5
mL/min, monitored at 220 nm. Reactions and
chromatography fractions were monitored by thin layer
chromatography (TLC) using Merck Kieselgel 60 F254
aluminium backed plates and visualised using a 254 nm UV
lamp and/or treatment with a suitable stain (phosphomolybdic
acid, ninhydrin or bromocresol green), followed by heating.
Flash chromatography was performed using silica gel (Davisil®
LC60 40-63 µm) as the stationary phase according to the
method of Still et al.63 All solvents used for flash
chromatography, including triethylamine, were distilled prior
to use except for acetic acid, which was analytical grade.
Eluent systems containing dichloromethane saturated with
ammonia were freshly prepared as follows: dichloromethane
(400 mL) and ammonium hydroxide (28%, 80 mL) were
shaken in a separating funnel and the dichloromethane layer
was separated, to which the required volume of methanol was
added if needed. All glassware used in moisture sensitive
reactions was oven dried, flame dried under vacuum and then
cooled under argon prior to use. All reactions were completed
at room temperature unless otherwise stated. Reaction progress
was monitored by thin layer chromatography (TLC) unless
otherwise noted. Purification of solvents and reagents was
carried out by the procedures described by Chai and
Armarego.64 All other solvents, reagents and starting materials
were purchased as reagent-grade from commercial sources and
used without further purification. All organic extracts were
dried over magnesium sulphate unless otherwise stated.
4.2.4 6-Amino-3-bromo-1-(4-methoxybenzyl)quinolin-2(1H)-
one (10). To a heterogeneous solution of the N-PMB protected
quinolinone 9 (1.50 g, 3.85 mmol) in MeOH (60 mL) and
AcOH (15 mL), was added Zn powder (2.51 g, 38.5 mmol)
and the reaction mixture was stirred for 30 min. The excess Zn
was filtered off and the filtrate was concentrated in vacuo. The
resulting residue was triturated with CH2Cl2, filtered again and
the filtrate washed with satd NaHCO3 (×2), brine (×2), dried,
filtered and the solvent was removed in vacuo to give a yellow
solid. The solid was purified by flash chromatography (60%
EtOAc/hexanes) and recrystallised from i-PrOH to give the
amine 10 (1.10 g, 80%) as a yellow solid; mp 168-170 °C; δH
(DMSO-d6): 8.35 (1H, s), 7.20 (1H, d, J 9.0 Hz), 7.14 (2H, d, J
9.0 Hz), 6.87 (1H, dd, J 9.0, 2.5 Hz), 6.86 (2H, d, J 9.0 Hz),
6.78 (1H, d, J 2.5 Hz), 5.43 (2H, s), 5.18 (2H, s), 3.71 (3H, s);
δC (DMSO-d6): 158.3, 156.8, 144.2, 140.8, 129.8, 128.6, 128.0,
121.3, 119.4, 116.2, 116.1, 114.0, 109.8, 55.0, 45.6; m/z (ESI):
4.2 Synthesis

6
359.0 (M[79Br]H+), 360.9 (M[81Br]H+); HRMS (ESI):
M[79Br]H+, found 359.0397. C17H16BrN2O2+ requires 359.0390.
°C for 4 h. The reaction mixture was cooled, diluted with 30%
NH4OH and extracted with EtOAc (×2). The combined organic
extracts were washed with brine (×4), dried, filtered and the
solvent was removed in vacuo to give a yellow solid. The solid
was purified by flash chromatography (0-1% MeOH/CH2Cl2)
and recrystallised from n-PrOH to give the nitrile 11c (220 mg,
82%) as a white solid; mp 202-205 °C; δH (DMSO-d6): 8.64
(1H, s), 8.29 (1H, d, J 2.0 Hz), 7.95 (1H, dd, J 9.0, 2.0 Hz),
7.62 (1H, d, J 9.0 Hz), 7.16 (2H, d, J 9.0 Hz), 6.87 (2H, d, J
9.0 Hz), 5.53 (2H, s), 3.70 (3H, s); δC (DMSO-d6): 158.5,
157.7, 140.9, 140.6, 133.3, 133.2, 128.0, 127.5, 120.4, 118.2,
118.1, 116.7, 114.2, 105.0, 55.0, 46.1; m/z (ESI): 761.0
(M2[79,81Br]Na+), 407.0 (M[79Br]K+), 409.0 (M[81Br]K+);
4.2.5
1-(3-Bromo-1-(4-methoxybenzyl)-2-oxo-1,2-
dihydroquinolin-6-yl)urea (11a). To a solution of amine 10
(1.00 g, 2.78 mmol) in AcOH (40 mL) was added NaOCN
(543 mg, 8.35 mmol) in H2O (10 mL) and the solution was
stirred at room temperature for 1 h. The reaction was poured
onto ice and the solid that separated was filtered and washed
with H2O. The solid was recrystallised from cyclohexanone
and n-PrOH to give the urea 11a (0.89 g, 80%) as a white
solid; mp 234-237 °C; δH (DMSO-d6): 8.69 (1H, br s), 8.53
(1H, s), 7.79 (1H, d, J 2.5 Hz), 7.53 (1H, dd, J 9.5, 2.5 Hz),
7.37 (1H, d, J 9.5 Hz), 7.16 (2H, d, J 8.5 Hz), 6.88 (2H, d, J
8.5 Hz), 5.89 (2H, s), 5.47 (2H, s), 3.70 (3H, s); δC (DMSO-
d6): 158.4, 157.2, 156.0, 141.3, 135.7, 133.1, 128.3, 128.0,
122.3, 120.6, 116.6, 115.72, 155.70, 114.0, 55.0, 45.8; m/z
(ESI): 402.0 (M[79Br]H+), 403.9 (M[81Br]H+); HRMS (ESI):
M[79Br]H+, found 402.0440. C18H17BrN3O3+ requires 402.0448.
HRMS
(ESI):
M2[79,81Br]Na+,
found
761.0206.
C36H26Br2N4NaO4+ requires 761.0193.
4.2.9
3-Bromo-1-(4-methoxybenzyl)-6-(3-
methoxyphenyl)quinolin-2(1H)-one (11d). To a solution of
iodo 12 (400 mg, 0.85 mmol) in 1,4-dioxane (20 mL) and H2O
(2 mL) was added 3-methoxyphenylboronic acid (155 mg, 1.02
mmol), Na2CO3 (180 mg, 1.70 mmol) and LiCl (72 mg, 1.70
mmol), and the mixture was degassed under argon. To this was
added Pd(PPh3)4 (50 mg, 5 mol%) and the reaction mixture
was heated to 100 °C overnight, then cooled and the solvent
was removed in vacuo. The resulting residue was dissolved in
CH2Cl2, washed with H2O (×2), brine (×2), dried, filtered and
the solvent was removed in vacuo to give a brown residue. The
residue was purified by flash chromatography (15-20%
EtOAc/hexanes) to give an inseperable mixture (10:1) of the
title compound 11d and the bis(3-methoxyphenyl) substituted
side-product, obtained as a light brown residue (199 mg, 52%).
On a small scale (approx. 200 mg), the impurity was removed
by flash chromatography (15% EtOAc/hexanes) to give the 6-
(3-methoxyphenyl) 11d as a white solid; mp 60-65 ºC; δH
(DMSO-d6): 8.65 (1H, s), 8.10 (1H, d, J 2.0 Hz), 7.90 (1H, dd,
J 9.0, 2.0 Hz), 7.54 (1H, d, J 9.0 Hz), 7.39 (1H, t, J 8.0 Hz),
7.25 (1H, d, J 8.0 Hz), 7.22 (1H, dd app t, J 2.0 Hz), 7.19 (2H,
d. J 8.5 Hz), 6.95 (1H, dd, J 8.0, 2.0 Hz), 6.88 (2H, d, J 8.5
Hz), 5.55 (2H, s), 3.82 (3H, s), 3.70 (3H, s); δC (DMSO-d6):
159.8, 158.4, 157.6, 141.7, 140.0, 137.7, 134.3, 130.1, 129.6,
128.2, 128.0, 126.3, 120.7, 118.8, 116.6, 116.0, 114.1, 113.2,
112.1, 55.2, 55.1, 45.9; m/z (ESI): 472.1 (M[79Br]Na+), 474.1
(M[81Br]Na+); HRMS (ESI): M[79Br]H+, found 450.0680.
C24H21BrNO3+ requires 450.0700.
4.2.6
N-(3-Bromo-1-(4-methoxybenzyl)-2-oxo-1,2-
dihydroquinolin-6-yl)acetamide (11b). To a solution of amine
10 (250 mg, 0.70 mmol) in anhydrous THF (10 mL) at 0 °C
was added Et3N (156 µL, 1.04 mmol) and AcCl (78 µL, 1.04
mmol), and the mixture was stirred for 5 min. The reaction
mixture was allowed to warm to room temperature and was
stirred for a further 30 min. The solvent was removed in vacuo,
H2O was added and the resulting solid was filtered to give the
acetanilide 11b (280 mg, 98%) as a tan solid; mp 133-136 °C;
δH (DMSO-d6): 10.11 (1H, s), 8.58 (1H, s), 8.01 (1H, d, J 2.5
Hz), 7.64 (1H, dd, J 9.0, 2.5 Hz), 7.44 (1H, d, J 9.0 Hz), 7.16
(2H, d, J 8.5 Hz), 6.87 (2H, d, J 8.5 Hz), 5.48 (2H, s), 3.69
(3H, s), 2.05 (3H, s); δC (DMSO-d6): 168.3, 158.4, 157.3,
141.3, 134.3, 134.2, 128.2, 128.1, 123.1, 120.4, 117.3, 116.8,
115.8, 114.1, 55.0, 45.8, 23.8; m/z (ESI): 423.0 (M[79Br]Na+),
425.0 (M[81Br]Na+); HRMS (ESI): M[79Br]H+, found
401.0483. C19H18BrN2O3+ requires 401.0496.
4.2.7 3-Bromo-6-iodo-1-(4-methoxybenzyl)quinolin-2(1H)-one
(12). To a suspension of amine 10 (2.20 g, 6.12 mmol) in conc.
HCl (60 mL) at -5°C, was added NaNO2 (845 mg, 12.2 mmol)
in H2O (40 mL) dropwise over 5 min and the reaction was
stirred for 1 h. The resulting yellow solution was added
dropwise to a -5°C solution of KI (50.0 g, 306.0 mmol) in H2O
(250 mL) and the reaction was stirred for 15 min at -5°C and
for a further 4 h at room temperature. The reaction mixture was
diluted with H2O and extracted with EtOAc (3×). The
combined organic extracts were washed with 2.5 M NaOH
(×2), 10% Na2S2O3 (×2), 2.5 M NaOH (×1), H2O (×1), brine
(×1), dried, filtered and the solvent was removed in vacuo to
give a light brown solid. The solid was recrystallised from n-
PrOH to give the iodo 12 (2.60 g, 91%) as a metallic brown
coloured solid; mp 202-205 °C; δH (DMSO-d6): 8.54 (1H, s),
8.14 (1H, d, J 2.0 Hz), 7.83 (1H, dd, J 9.0, 2.0 Hz), 7.28 (1H,
d, J 9.0 Hz), 7.13 (2H, d, J 8.5 Hz), 6.86 (2H, d, J 8.5 Hz),
5.47 (2H, s), 3.70 (3H, s). δC (DMSO-d6): 158.4, 157.4, 140.3,
138.9, 137.8, 136.2, 127.9, 127.8, 122.4, 117.7, 117.3, 114.1,
86.4, 55.0, 45.8; m/z (ESI): 507.9 (M[79Br]K+), 509.9
(M[81Br]K+); HRMS (ESI): M[79Br]H+, found 469.9251.
C17H15BrINO2+ requires 469.9248.
4.2.10 3-Bromo-6-ethoxy-1-(4-methoxybenzyl)quinolin-2(1H)-
one (11e). To a suspension of iodo 12 (600 mg, 1.28 mmol),
Cs2CO3 (832 mg, 2.55 mmol) and 1,10-phenanthroline (46 mg,
20 mol%) in EtOH (20 mL) was added CuI (24 mg, 10 mol%)
and the mixture was heated to 110 °C in a pressure tube
overnight. The reaction mixture was cooled, passed through a
silica pad eluting with EtOAc and the filtrate was concentrated
in vacuo to give the crude material as a 1:1:1 mixture of the
desired ethoxy 11e, as well as two other cross-coupled
side-products. The mixture was separated by flash
chromatography (15% EtOAc/hexanes) to give 6-ethoxy 11e
(170 mg, 35%) as a tan solid; mp 118-121 °C; δH (DMSO-d6):
8.51 (1H, s), 7.39 (1H, d, J 9.5 Hz), 7.30 (1H, d, J 3.0 Hz),
7.19 (1H, dd, J 9.5, 3.0 Hz), 7.15 (2H, d, J 9.0 Hz), 6.87 (2H,
d, J 9.0 Hz), 5.48 (2H, s), 4.04 (2H, q, J 7.0 Hz), 3.69 (3H, s),
1.33 (3H, t, J 7.0 Hz); δC (DMSO-d6): 158.4, 157.2, 153.7,
141.0, 132.7, 128.3, 128.0, 121.1, 119.9, 116.9, 116.7, 114.1,
110.8, 63.5, 55.0, 45.9, 14.5; m/z (ESI): 426.0 (M[79Br]K+),
428.0 (M[81Br]K+); HRMS (ESI): M[79Br]H+, found 388.0548.
C19H19BrNO3+ requires 388.0543.
4.2.8 3-Bromo-6-cyano-1-(4-methoxybenzyl)quinolin-2(1H)-
one (11c). To a solution of iodo 12 (340 mg, 0.72 mmol) in
DMF (10 mL) was added Zn(CN)2 (55 mg, 0.47 mmol) and the
mixture was purged with argon. To this was added Pd(PPh3)4
(62 mg, 7.5 mol%) and the reaction mixture was heated to 90

7
Side-products 3-Ethoxy-6-iodo-1-(4-methoxybenzyl)quinolin-
2(1H)-one (13a) and 3,6-diethoxy-1-(4-
methoxybenzyl)quinolin-2(1H)-one (13b) were obtained from
the crude mixture using flash chromatography (60-80%
EtOAc/hexanes) to give:
Hz), 7.21-7.17 (3H, m), 7.08 (1H, dd, J 1.5 Hz), 6.87 (2H, d,
J 8.5 Hz), 6.83 (1H, dd app t, J 1.5 Hz), 5.79 (2H, s), 5.53 (2H,
s), 3.70 (3H, s), 3.39-3.37 (4H, m), 3.34 (2H, s), 3.15 (2H, q, J
6.0 Hz), 2.30 (2H, t, J 6.5 Hz), 2.28 (4H, br s), 1.87-1.84 (4H,
m), 1.44-1.40 (4H, m), 1.32-1.28 (2H, m); δC (DMSO-d6):
169.8, 158.3, 157.6, 156.0, 153.9, 141.3, 140.6, 137.2, 135.6,
132.5, 128.7, 128.0, 127.6, 121.9, 117.0, 115.1, 114.2, 114.0,
108.8, 107.4, 57.6, 55.0, 54.0, 45.7, 45.1, 42.8, 36.4, 25.5,
25.0, 24.0; m/z (ESI): 695.4 (MH+); HRMS (ESI): MH+, found
695.3667. C38H47N8O5+ requires 695.3664.
3-Ethoxy-6-iodo 13a (70 mg) as a light brown solid; mp 184-
190 °C; δH (DMSO-d6): 8.12 (1H, d, J 2.0 Hz), 7.79 (1H, dd, J
9.0, 2.0 Hz), 7.19 (1H, d, J 9.0 Hz), 7.10 (2H, d, J 9.0 Hz),
6.85 (2H, d, J 9.0. Hz), 6.13 (1H, s), 5.35 (2H, br s), 4.22 (2H,
q, J 7.0 Hz), 3.69 (3H, s), 1.44 (3H, t, J 7.0 Hz); δC (DMSO-
d6): 162.3, 159.9, 158.2, 139.2, 138.2, 130.8, 128.6, 127.7,
118.0, 117.6, 114.0, 97.3, 85.2, 64.7, 55.0, 43.5, 14.0; m/z
(ESI): 436.0 (MH+); HRMS (ESI): MH+, found 436.0388.
C19H19INO3+ requires 436.0405.
4.2.13
1-(1-(4-Methoxybenzyl)-2-oxo-3-(phenylamino)-1,2-
dihydroquinolin-6-yl)urea (16c). Bromo compound 11a (240
mg, 0.60 mmol) was coupled with aniline according to General
Procedure A, except that once the reaction was complete, the
solvent was removed in vacuo and H2O was added. The
resulting precipitate was filtered and purified by flash
chromatography (2.5-4.0% MeOH/CH2Cl2) followed by
recrystallisation from n-BuOH to give the N-PMB protected
quinolinone 16c (105 mg, 44%) as a grey solid; mp >300 °C;
δH (DMSO-d6): 8.46 (1H, s), 8.03 (1H, s), 7.67 (1H, d, J 2.5
Hz), 7.42-7.35 (5H, m), 7.41 (1H, d, J 9.0 Hz), 7.20 (2H, d, J
9.0 Hz), 7.18 (1H, dd, J 9.0, 2.0 Hz), 7.00 (1H, tt, J 7.0, 1.0
Hz), 6.88 (2H, d, J 9.0 Hz), 5.83 (2H, s), 5.53 (1H, s), 3.70
(3H, s); δC (DMSO-d6): 158.3, 157.6, 156.1, 141.1, 135.6,
132.4, 129.2, 128.7, 128.1, 127.8, 121.9, 121.7, 119.6, 117.2,
115.0, 114.3, 114.0, 107.6, 55.0, 45.1; m/z (ESI): 437.2
3,6-Diethoxy 13b (45 mg) as a light yellow solid; mp 131-135
°C; δH (DMSO-d6): 7.31 (1H, s), 7.30 (1H, d, J 9.0 Hz), 7.15
(1H, dd, J 9.0, 3.0 Hz), 7.12 (2H, d, J 9.0 Hz), 6.85 (2H, d, J
9.0 Hz), 6.10 (1H, s), 5.36 (2H, br s), 4.22 (2H, q, J 7.0 Hz),
4.21 (2H, q, J 7.0 Hz), 3.69 (3H, s), 1.44 (3H, t, J 7.0 Hz), 1.31
(3H, t, J 7.0 Hz). δC (DMSO-d6): 162.2, 160.6, 158.2, 153.2,
133.0, 129.1, 127.8, 119.6, 166.64, 116.59, 113.9, 105.8, 96.9,
64.3, 63.5, 55.0, 43.5, 14.6, 14.0; m/z (ESI): 354.1 (MH+);
+
HRMS (ESI): MH+, found 354.1688. C21H24NO4 requires
354.1700.
+
4.2.11 General Procedure A. Preparation of N-PMB protected
quinolinones via Palladium Catalysed Buchwald-Hartwig
Cross-Coupling. N-(3-((1-(4-Methoxybenzyl)-2-oxo-6-ureido-
1,2-dihydroquinolin-3-yl)amino)phenyl)pyrrolidine-1-
(MNa+); HRMS (ESI): MH+, found 415.1753. C24H23N4O3
requires 415.1765.
4.2.14
N-(3-((6-Acetamido-1-(4-methoxybenzyl)-2-oxo-1,2-
carboxamide (16a). A mixture of bromo compound 11a (400
mg, 0.99 mmol), custom aniline 14 (245 mg, 1.19 mmol) and
K2CO3 (302 mg, 2.18 mmol) in anhydrous t-BuOH (40 mL)
was degassed under argon. To this was added XPhos (47 mg,
10 mol%) and Pd2(dba)3 (46 mg, 5 mol%) and the reaction
mixture was heated to reflux overnight, then allowed to cool to
room temperature. To the reaction mixture was added H2O and
the mixture was extraced with CH2Cl2 (×2). The combined
organic extracts were washed with brine (×2), dried, filtered
and the solvent removed in vacuo to give a dark red residue.
The residue was purified by flash chromatography (2-4%
MeOH/CH2Cl2) and recrystallised from n-PrOH to give the N-
PMB protected quinolinone 16a (266 mg, 51%) as a beige
solid; mp 214-217 °C; δH (DMSO-d6): 8.50 (1H, s), 8.12 (1H,
s), 7.87 (1H, s), 7.65 (1H, dd, J 2.5, 1.5 Hz), 7.63 (1H, d, J 2.5
Hz), 7.36 (1H, s), 7.28 (1H, d, J 9.5 Hz), 7.22-7.19 (5H, m),
6.94-6.92 (1H, m), 6.88 (2H, d, J 9.5 Hz), 5.81 (2H, s), 5.54
(2H, s), 3.71 (3H, s), 3.40-3.37 (4H, m), 1.87-1.84 (4H, m); δC
(DMSO-d6): 158.3, 157.6, 156.0, 153.9, 141.4, 140.9, 135.6,
132.4, 129.0, 128.7, 128.0, 127.7, 122.0, 117.1, 115.1, 114.2,
114.0, 113.5, 113.2, 110.2, 107.5, 55.0, 45.7, 45.1, 25.0; m/z
(ESI): 527.2 (MH+). HRMS (ESI): MH+, found 527.2380.
C29H31N6O4+ requires 527.2402.
dihydroquinolin-3-yl)amino)phenyl)pyrrolidine-1-carboxamide
(16d). Bromo compound 11b (250 mg, 0.62 mmol) was
coupled with custom aniline 14 according to General
Procedure A. Purification by flash chromatography (2.5%
MeOH/CH2Cl2) and recrystallisation from n-PrOH afforded the
N-PMB protected quinolinone 16d (265 mg, 81%) as an off-
white solid; mp 168-172 °C; δH (DMSO-d6): 9.96 (1H, s), 8.13
(1H, s), 7.93 (1H, s), 7.81 (1H, dd app t, J 1.5 Hz), 7.66 (1H,
dd app t, J 2.0 Hz), 7.36-7.34 (3H, m), 7.21-7.17 (4H, m),
6.94-6.92 (1H, m), 6.87 (2H, d, J 8.5 Hz), 5.54 (2H, s), 3.70
(3H, s), 3.40-3.37 (4H, m), 2.03 (3H, s), 1.87-1.85 (4H, m); δC
(DMSO-d6): 168.6, 158.8, 158.2, 154.4, 141.9, 141.3, 134.8,
133.1, 129.5, 129.2, 129.1, 128.5, 122.3, 118.3, 116.2, 115.6,
114.5, 114.2, 113.7, 110.8, 107.7, 55.5, 46.2, 45.6, 25.5, 24.4;
m/z (ESI): 548.3 (MNa+); HRMS (ESI): MH+, found 526.2431.
C30H32N5O4+ requires 526.2449.
4.2.15
N-(3-((6-Cyano-1-(4-methoxybenzyl)-2-oxo-1,2-
dihydroquinolin-3-yl)amino)phenyl)pyrrolidine-1-carboxamide
(16e). Bromo compound 11c (175 mg, 0.47 mmol) was
coupled with custom aniline 14 according to General
Procedure A. Purification by flash chromatography (1-2%
MeOH/CH2Cl2) and recrystallisation from n-PrOH afforded the
N-PMB protected quinolinone 16e (215 mg, 92%) as a yellow
solid; mp 204-206 °C; δH (DMSO-d6): 8.15 (1H, s), 8.11 (1H,
s), 8.05 (1H, d, J 2.0 Hz), 7.63-7.60 (2H, m), 7.53 (1H, d, J 9.0
Hz), 7.45 (1H, s), 7.24-7.22 (2H, m), 7.19 (2H, d, J 8.5 Hz),
6.97-6.95 (1H, m), 6.89 (2H, d, J 8.5 Hz), 5.60 (2H, s), 3.70
(3H, s), 3.40-3.37 (4H, m), 1.87-1.84 (4H, m); δC (DMSO-d6):
158.5, 158.2, 153.9, 141.4, 140.2, 135.3, 134.0, 130.5, 129.0,
128.0, 127.93, 127.91, 122.5, 118.9, 115.9, 114.4, 114.2, 114.1,
111.7, 105.6, 104.9, 55.0, 45.7, 45.3, 25.0; m/z (ESI): 516.3
4.2.12
N-(3-((1-(4-Methoxybenzyl)-2-oxo-6-ureido-1,2-
dihydroquinolin-3-yl)amino)-5-(2-oxo-2-((2-(piperidin-1-
yl)ethyl)amino)ethyl)phenyl)pyrrolidine-1-carboxamide (16b).
Bromo compound 11a (350 mg, 0.87 mmol) was coupled with
custom aniline 15 according to General Procedure A.
Purification by flash chromatography (5-6% MeOH/CH2Cl2
satd with NH3) and recrystallisation from EtOH afforded the
N-PMB protected quinolinone 16b (350 mg, 58%) as a white
solid; mp 156-159 °C; δH (DMSO-d6): 8.50 (1H, s), 8.12 (1H,
s), 7.81 (1H, t, J 5.5 Hz), 7.76 (1H, s), 7.60 (1H, d, J 2.5 Hz),
7.50 (1H, dd app t, J 2.0 Hz), 7.32 (1H, s), 7.27 (1H, d, J 9.0
+
(MNa+); HRMS (ESI): MH+, found 494.2164. C29H28N5O3
requires 494.2187.

8
4.2.16 N-(3-((1-(4-Methoxybenzyl)-6-(3-methoxyphenyl)-2-
solid that separated was filtered and the filtrate was extracted
with CH2Cl2 (×3). The combined organic extracts were washed
with brine (×2), dried, filtered and the solvent was removed in
vacuo to give a beige solid. The resulting crude solid was
combined with the previously isolated solid and purified by
flash chromatography (5-6% MeOH/CH2Cl2 satd with NH3)
followed by recrystallisation from EtOH to give the title
compound 1a (65 mg, 65%) as a white solid; mp >300°C; δH
(DMSO-d6): 11.96 (1H, s), 8.47 (1H, s), 8.11 (1H, s), 7.71 (1H,
s), 7.61 (1H, s), 7.59 (1H, d, J 2.0 Hz), 7.32 (1H, s), 7.21-7.14
(4H, m), 6.92-6.88 (1H, m), 5.79 (2H, s), 3.40-3.37 (4H, m),
1.87-1.84 (4H, m); δC (DMSO-d6): 157.7, 156.1, 153.9, 141.4,
140.9, 135.3, 133.0, 128.9, 127.3, 121.1, 117.2, 114.8, 113.4,
113.2, 113.0, 109.9, 107.4, 45.7, 25.0; m/z (ESI): 407.2 (MH+);
oxo-1,2-dihydroquinolin-3-yl)amino)phenyl)pyrrolidine-1-
carboxamide (16f). Bromo compound 11d (150 mg, 0.33
mmol) was coupled with custom aniline 14 according to
General Procedure A. Purification by flash chromatography (0-
2% MeOH/CH2Cl2) afforded the N-PMB protected quinolinone
16f (140 mg, 73%) as a yellow solid; mp 103-106 °C; δH
(DMSO-d6): 8.11 (1H, s), 7.94 (1H, s), 7.88 (1H, d, J 2.0 Hz),
7.63 (1H, s), 7.57 (1H, dd, J 9.0, 2.0 Hz), 7.56 (1H, s), 7.45
(1H, d, J 9.0 Hz), 7.35 (1H, t, J 8.0 Hz), 7.25 (1H, d, J 8.0 Hz),
7.23-7.21 (5H, m), 6.99-6.96 (1H, m), 6.93-6.89 (3H, m), 5.61
(2H, br s), 3.81 (3H, s), 3.70 (3H, s), 3.39-3.36 (4H, m), 1.86-
1.84 (4H, m); δC (DMSO-d6):159.8, 158.4, 158.1, 153.9, 141.4,
140.9, 140.8, 134.3, 132.8, 132.3, 129.9, 129.0, 128.5, 128.0,
124.29, 124.27, 122.2, 118.8, 115.3, 114.1, 113.8, 113.5,
112.9, 111.9, 111.0, 107.7, 55.11, 55.05, 45.7, 45.2, 25.0; m/z
(ESI): 597.3 (MNa+); HRMS (ESI): MH+, found 575.2631.
C35H35N4O4+ requires 575.2653.
+
HRMS (ESI): MH+, found 407.1826. C21H23N6O3 requires
407.1827.
4.2.20 N-(3-(2-Oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)-5-
((2-oxo-6-ureido-1,2-dihydroquinolin-3-
4.2.17
N-(3-((6-Ethoxy-1-(4-methoxybenzyl)-2-oxo-1,2-
yl)amino)phenyl)pyrrolidine-1-carboxamide (1b). N-PMB
protected quinolinone 16b (160 mg, 0.23 mmol) was treated
according to General Procedure B, except that the crude
product was extracted using 15% i-PrOH in CH2Cl2.
Purification by flash chromatography (7-8% MeOH/CH2Cl2
satd with NH3) afforded an off-white solid (86 mg, 65%).
Further purification by preparatory reverse phase HPLC (20-
60% MeCN/10 mM aq NH4CO3) provided the title compound
1b (35 mg, 26%) as a white solid; mp >300 °C; δH (DMSO-d6):
11.88 (1H, s), 8.41 (1H, s), 8.04 (1H, s), 7.74 (1H, t, J 5.0 Hz),
7.56 (1H, d, J 2.5 Hz), 7.53 (1H, s), 7.46 (1H, dd app t, J 2.0
Hz), 7.27 (1H, s), 7.19 (1H, dd, J 9.0, 2.0 Hz), 7.15 (1H, d, J
9.0 Hz), 7.08 (1H, dd app t, J 2.0 Hz), 6.79 (1H, dd app t, J 2.0
Hz), 5.70 (2H, s), 3.40-3.37 (4H, m), 3.37 (2H, s), 3.21-3.17
(2H, m), 2.38 (6H, br s), 1.87-1.85 (4H, m), 1.48-1.43 (4H, m),
1.36-1.31 (2H, m); δC (DMSO-d6): 169.9, 157.5, 156.0, 153.8,
141.2, 140.6, 137.0, 135.2, 133.0, 127.3, 121.0, 117.2, 114.8,
113.9, 113.8, 113.5, 108.3, 107.4, 57.2, 53.7, 45.6, 42.7, 36.1,
25.1, 24.9, 23.6; m/z (ESI): 575.3 (MH+); HRMS (ESI): MH+,
found 575.3087. C30H39N8O4+ requires 575.3089.
dihydroquinolin-3-yl)amino)phenyl)pyrrolidine-1-carboxamide
(16g). Bromo compound 11e (142 mg, 0.37 mmol) was
coupled with custom aniline 14 according to General
Procedure A. Purification by flash chromatography (70%
EtOAc/hexanes) afforded the N-PMB protected quinolinone
16g (145 mg, 78%) as a white solid; mp 201-203 °C; δH
(DMSO-d6): 8.10 (1H, s), 7.88 (1H, s), 7.59 (1H, dd app t, J
2.0 Hz), 7.40 (1H, s), 7.29 (1H, d, J 9.5 Hz), 7.21-7.17 (4H,
m), 7.08 (1H, d, J 3.0 Hz), 6.95-6.93 (1H, m), 6.88 (2H, d, J
9.0 Hz), 6.85 (1H, dd, J 9.0, 3.0 Hz), 5.54 (2H, s), 4.01 (2H, q,
J 7.0 Hz), 3.70 (3H, s), 3.39-3.37 (4H, m), 1.87-1.84 (4H, m),
1.31 (3H, t, J 7.0 Hz); δC (DMSO-d6): 158.3, 157.6, 154.0,
153.9, 141.4, 140.8, 132.8, 129.0, 128.7, 128.0, 127.1, 122.8,
116.0, 114.3, 114.0, 113.7, 113.5, 111.0, 109.3, 107.2, 65.2,
55.0, 45.7, 45.1, 25.0, 14.7; m/z (ESI): 535.3 (MNa+); HRMS
(ESI): MH+, found 513.2483. C30H33N4O4+ requires 513.2497.
4.2.18 N-(3-((1-(4-methoxybenzyl)-2-oxo-6-(2H-tetrazol-5-yl)-
1,2-dihydroquinolin-3-yl)amino)phenyl)pyrrolidine-1-
carboxamide (16h). To a suspension of N-PMB protected
quinolinone 16e (220 mg, 0.44 mmol) and TMS-N3 (586 µL,
4.46 mmol) under argon was added TBAF (1M in
tetrahydrofuran containing 5 wt. % of H2O, 534 µL, 0.53
mmol) and the mixture was heated to 85 °C for 48 h. The
reaction was cooled, concentrated in vacuo and diluted out
with 1M HCl. The solid that separated was filtered, washed
with 1M HCl and H2O to give the tetrazole intermediate 16h
(234 mg, 98%) as a brown solid; mp 156-162 °C; δH (DMSO-
d6): 8.23 (1H, d, J 2.0 Hz), 8.12 (1H, s), 8.08 (1H, br s), 7.86
(1H, dd, J 9.0, 2.0 Hz), 7.68 (1H, dd, J 2.0, 1.0 Hz), 7.63 (1H,
d, J 9.0 Hz), 7.53 (1H, s), 7.24-7.21 (4H, m), 6.98 (1H, m),
6.89 (2H, d, J 9.0 Hz), 5.63 (2H, s), 3.70 (3H, s), 3.41-3.38
(4H, m), 1.88-1.85 (4H, m); δC (DMSO-d6): 158.4, 158.1,
153.9, 141.4, 140.4, 134.3, 133.4, 129.0, 128.2, 128.0, 124.9,
123.7, 122.3, 115.8, 114.13, 114.09, 113.7, 111.0, 106.6, 55.0,
45.7, 45.3, 25.0; m/z (ESI): 535.4 (MH-); HRMS (ESI): MH+,
found 537.2334. C29H29N8O3+ requires 537.2358.
4.2.211-(2-Oxo-3-(phenylamino)-1,2-dihydroquinolin-6-
yl)urea (1c). N-PMB protected quinolinone 16c (80 mg, 0.19
mmol) was treated according to General Procedure B, except
once quenched with satd NaHCO3 the mixture was refrigerated
overnight and the resulting precipitate was collected by
filtration. Purification by flash chromatography (5-20%
MeOH/CH2Cl2) and washing with H2O and acetone afforded
the title compound 1c (26 mg, 47%) as a light brown solid; mp
>300 °C; δH (DMSO-d6): 11.96 (1H, s), 8.41 (1H, s), 7.87 (1H,
s), 7.61 (1H, d, J 2.0 Hz), 7.38-7.32 (5H, m), 7.17 (1H, dd, J
9.0, 2.0 Hz), 7.13 (1H, d, J 9.0 Hz), 6.97 (1H, tt, J 7.0, 1.0 Hz),
5.79 (2H, s); δC (DMSO-d6): 157.7, 156.1, 141.1, 135.3, 133.0,
129.2, 127.5, 121.5, 121.1, 119.3, 117.3, 114.9, 113.6, 107.6;
m/z (ESI): 293.1 (MH-); HRMS (ESI): MH+, found 295.1191.
C16H15N4O2+ requires 295.1190.
4.2.22
N-(3-((6-Acetamido-2-oxo-1,2-dihydroquinolin-3-
yl)amino)phenyl)pyrrolidine-1-carboxamide (1d). N-PMB
protected quinolinone 16d (115 mg, 0.22 mmol) was treated
according to General Procedure B. Purification by flash
chromatography (5-7.5% MeOH/CH2Cl2) and recrystallisation
from n-PrOH afforded the title compound 1d (54 mg, 62%) as
a white solid; mp 231-234 °C; δH (DMSO-d6): 12.10 (1H, s),
9.91 (1H, s), 8.11 (1H, s), 7.78 (1H, d, J 2.0 Hz), 7.75 (1H, s),
7.62 (1H, s), 7.34 (1H, dd, J 9.0, 2.0 Hz), 7.31 (1H, s), 7.19-
7.16 (3H, m), 6.90-6.88 (1H, m), 3.40-3.37 (4H, m), 2.03 (3H,
4.2.19 General Procedure B. Preparation of 3-Anilino-
quinolin-2-(1H)-ones via Acid Catalysed Debenzylation. N-(3-
((2-Oxo-6-ureido-1,2-dihydroquinolin-3-
yl)amino)phenyl)pyrrolidine-1-carboxamide (1a). To
a
solution of N-PMB protected quinolinone 16a (140 mg, 0.27
mmol) in TFA (5 mL) was added TfOH (82 µL, 0.93 mmol).
The reaction mixture was stirred at room temperature for 3h
and then carefully poured onto ice-cold satd NaHCO3. The

9
s), 1.87-1.84 (4H, m); δC (DMSO-d6): 168.0, 157.8, 153.9,
141.4, 140.9, 134.0, 133.2, 129.0, 128.4, 121.0, 117.9, 115.0,
114.9, 113.4, 113.1, 109.0, 107.2, 45.7, 25.0, 23.9; m/z (ESI):
428.2 (MNa+); HRMS (ESI): MH+, found 406.1879.
C22H24N5O3+ requires 406.1874.
7.66 (1H, dd app t, J 2.0 Hz), 7.52 (1H, s), 7.27 (1H, d, J 8.5
Hz), 7.20 (1H, dt, J 7.5, 2.0 Hz), 7.16 (1H, t, J 8.0 Hz), 6.91
(1H, dt, J 7.5, 2.0 Hz), 3.42-3.39 (4H, m), 1.88-1.85 (4H, m);
δC (DMSO-d6): 160.4, 158.1, 153.9, 141.4, 141.0, 132.6, 131.5,
128.9, 127.2, 123.9, 122.2, 120.9, 114.6, 113.1, 112.7, 109.4,
108.1, 45.7, 25.0; m/z (ESI): 415.3 (MH-); HRMS (ESI): MH+,
found 417.1786. C21H21N8O2+ requires 417.1782.
4.2.23
N-(3-((6-Cyano-2-oxo-1,2-dihydroquinolin-3-
yl)amino)phenyl)pyrrolidine-1-carboxamide (1e). N-PMB
protected quinolinone 16e (200 mg, 0.41 mmol) was treated
according to General Procedure B. Purification by flash
chromatography (2.5-5% MeOH/CH2Cl2) and recrystallisation
from EtOH afforded the title compound 1e (44 mg, 29%) as a
beige solid; mp 279-281 °C; δH (DMSO-d6): 12.47 (1H, s),
8.00 (1H, s), 7.99 (1H, s), 7.98 (1H, d, J 2.0 Hz), 7.60 (1H, dd,
J 8.0, 2.0 Hz), 7.58 (1H, dd app t, J 2.0 Hz), 7.37 (1H, s), 7.36
(1H, d, J 8.5 Hz), 7.22-7.18 (2H, m), 6.94-6.91 (1H, m), 3.39-
3.37 (4H, m), 1.87-1.84 (4H, m); δC (DMSO-d6): 158.3, 153.9,
141.4, 140.3, 135.1, 134.7, 129.9, 129.0, 127.8, 121.6, 119.3,
115.6, 114.2, 114.0, 111.5, 105.5, 104.3, 45.7, 25.0; m/z (ESI):
396.1 (MNa+); HRMS (ESI): MH+, found 374.1617.
C21H20N5O2+ requires 374.1612.
4.3 Biological Screening
All compounds were assayed using a radiometric in vitro
kinase assay that was in accordance of Biondi et al.66, except
magnesium acetate was used instead of magnesium chloride.
Compounds tested using the biological assay were prepared in
freshly distilled dimethyl sulfoxide to a stock concentration of
100 mM and then diluted as required for assays. The literature
inhibitor compound RM1 was used as the positive control (IC50
of 200 nM).17 PDK1 enzyme was purchased from Invitrogen at
a purity of 70% and diluted to a concentration of 6.25 ng/µL
using 50 mM Tris buffer (adjusted to pH 7.5) containing 0.1%
β-mercaptoethanol and 1% bovine serum albumin. PDKtide
was purchased from GL Biochem Pty Ltd at a purity of 95.3%
by RP-HPLC analysis and used without further purification.
P81 cellulose paper was purchased from Millipore as pre-
labelled squares and stored in the freezer at -5 ºC. [γ-32P]-ATP
was purchased from Perkin Elmer Easytides at a 10 mCi/mL
concentration and was stored at 2-8 ºC. 32P radiation was
counted using Pharmcia Wallac 1410 Liquid Scintillation
Counter using “easy count” mode. All other reagents used in
the assay were purchased from Sigma-Aldrich and used
without further purification.
4.2.24 N-(3-((6-(3-Methoxyphenyl)-2-oxo-1,2-dihydroquinolin-
3-yl)amino)phenyl)pyrrolidine-1-carboxamide (1f). N-PMB
protected quinolinone 16f (120 mg, 0.21 mmol) was treated
according to General Procedure B. Purification by flash
chromatography (2.5% MeOH/CH2Cl2) and recrystallisation
from n-PrOH afforded the title compound 1f (51 mg, 55%) as a
white solid; mp 203-206 °C; δH (DMSO-d6): 12.18 (1H, s),
8.09 (1H, s), 7.81 (1H, d, J 2.0 Hz), 7.78 (1H, s), 7.59 (1H, s),
7.58 (1H, dd, J 8.5, 2.0 Hz), 7.48 (1H, s), 7.36 (1H, t, J 8.0
Hz), 7.33 (1H, d, J 8.5 Hz), 7.25 (1H, ddd, J 8.0, 2.0, 1.0 Hz),
7.22-7.21 (1H, m), 7.20-7.18 (2H, m), 6.95-6.92 (1H, m), 6.91
(1H, ddd, J 8.0, 2.0, 1.0 Hz), 3.83 (3H, s), 3.39-3.36 (4H, m),
1.86-1.84 (4H, m); δC (DMSO-d6): 159.7, 158.1, 153.9, 141.41,
141.39, 140.8, 134.0, 133.4, 132.0, 129.9, 128.9, 124.2, 123.4,
121.4, 118.8, 115.2, 113.5, 113.4, 112.6, 111.9, 110.7, 107.6,
55.1, 45.7, 25.0; m/z (ESI): 477.2 (MNa+); HRMS (ESI): MH+,
found 455.2086. C27H27N4O3+ requires 455.2078.
Acknowledgements
The authors would like to thank Dr. Susanne Cutts (La
Trobe Institute for Molecular Science, La Trobe University)
for her assistance with the biological assays and Dr. Jason
Dang (Monash Institute of Pharmaceutical Sciences, Monash
University) for HRMS analysis. N.J O’Brien would like to
thank the Australian government for financial support through
the provision of an Australian Postgraduate Awards (APA)
scholarship.
4.2.25
N-(3-((6-Ethoxy-2-oxo-1,2-dihydroquinolin-3-
yl)amino)phenyl)pyrrolidine-1-carboxamide (1g). N-PMB
protected quinolinone 16g (120 mg, 0.23 mmol) was treated
according to General Procedure B. Purification by flash
chromatography (5% MeOH/CH2Cl2) and recrystallisation
from EtOH afforded the title compound 1g (55 mg, 60%) as a
white solid; mp 225-229 °C; δH (DMSO-d6): 11.98 (1H, s),
8.08 (1H, s), 7.72 (1H, s), 7.56 (1H, dd app t, J 1.5 Hz), 7.34
(1H, s), 7.18-7.16 (3H, m), 7.01 (1H, d, J 2.5 Hz), 6.92-6.89
(1H, m), 6.86 (1H, dd, J 9.0, 2.5 Hz), 4.01 (2H, q, J 7.0 Hz),
3.39-3.36 (4H, m), 1.87-1.84 (4H, m), 1.32 (3H, t, J 7.0 Hz);
δC (DMSO-d6): 157.5, 153.8, 141.4, 140.8, 133.4, 128.9, 126.6,
121.8, 115.8, 114.5, 113.4, 113.3, 110.6, 108.2, 107.2, 63.2,
45.6, 25.0, 14.7; m/z (ESI): 415.2 (MNa+); HRMS (ESI): MH+,
found 393.1926. C22H25N4O3+ requires 393.1922.
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