Synthesis and biological evaluation of 2-anilino-4-substituted-7H- pyrrolopyrimidines as PDK1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.06.018

PDK1, a biological target that has attracted a large amount of attention recently, is responsible for the positive regulation of the PI3K/Akt pathway that is often activated in a large number of human cancers. A series of second-generation 2-anilino-4-substituted-7H-pyrrolopyrimidines were synthesised by installation of various functions at the 4-position of the 7H-pyrrolopyrimidine scaffold. All compounds were screened against the isolated PDK1 enzyme and dose response analysis was obtained on the best compounds of the series. Crown Copyright

Full text of DOI:10.1016/j.bmc.2014.06.018

Bioorganic & Medicinal Chemistry 22 (2014) 3879–3886
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 2-anilino-4-substituted-7H-
pyrrolopyrimidines as PDK1 inhibitors
⇑
Nathan J. O’Brien, Martin Brzozowski, Melissa J. Buskes, Leslie W. Deady, Belinda M. Abbott
Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Vic 3086, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
PDK1, a biological target that has attracted a large amount of attention recently, is responsible for the
positive regulation of the PI3K/Akt pathway that is often activated in a large number of human cancers.
Received 30 April 2014
Revised 30 May 2014
Accepted 8 June 2014
Available online 18 June 2014
A
series of second-generation 2-anilino-4-substituted-7H-pyrrolopyrimidines were synthesised by
installation of various functions at the 4-position of the 7H-pyrrolopyrimidine scaffold. All compounds
were screened against the isolated PDK1 enzyme and dose response analysis was obtained on the best
compounds of the series.
Keywords:
3-Phosphoinositide-dependent kinase 1
(PDK1)
Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
2-Anilino-4-substituted-pyrrolopyrimidines
Inhibitor
Suzuki cross-coupling
Buchwald–Hartwig cross-coupling
1. Introduction
Recent experimental evidence has shown that specific
inhibitors of this pathway can promote apoptosis in cells and
3-Phosphoinositide-dependent kinase 1 (PDK1) is a biological
target that has attracted a considerable amount of interest in
recent years, within both academia and industry, for the develop-
ment of oncology therapeutics.1–3 PDK1 is the master regulator of
the AGC kinase signal transduction and plays a crucial role in the
positive regulation of the PI3K/Akt signalling pathway, which is
active in many human cancers.4,5 PDK1 is critical for the regulation
for as many as 23 different agonist stimulated AGC kinases
including Akt,6,7 PKC isoforms,8 P70 S6K,9 p90 SGK10 and RSK11
by phosphorylation at its activation loop. Its activity is modulated
by phosphatidylinositol 3-kinase (PI3K) through the stimulation of
the secondary messenger, phosphatidylinositol 3,4,5-triphosphate
(PIP3).
reduce tumour growth.16 As PDK1 plays a key regulatory role
within the PI3K/Akt signalling pathway, targeted inhibition of this
kinase provides an attractive target for the treatment of cancer.1,14
Recently, we reported the synthesis of a first-generation series
of novel substituted 2-anilino-7H-pyrrolopyrimidines17 that
contained varied functionality in the 2-anilino substituent, for
example, compound 1 (Fig. 1). These compounds were designed
by a molecular modelling approach in which the core of the
inhibitor was bound to the ATP binding site of PDK1, and the
additional functionality was an attempt to gain potency and
selectivity (Fig. 2). Biological screening against isolated PDK1 was
undertaken, but only modest inhibition was observed.
PIP3 interacts with several downstream effector proteins that
control the activity and subcellular localisation of many important
signal transducers that are vital to cell growth, metabolism,
proliferation and survival.4,12,13 Overstimulation of PIP3, by
disruption or dysregulation of the PI3K/Akt pathway through gene
mutation, leads to constitutive activation of downstream kinases
and subsequent promotion of tumour proliferation, reduced
apoptosis and angiogenesis.14,15
Figure 1. Previously synthesised target compound 117 with 4-methoxyphenyl-9H-
purine 2 inhibitor reported by Blanchard et al.18
⇑
Corresponding author. Tel.: +61 3 94792520; fax: +61 3 94791266.
E-mail address: b.abbott@latrobe.edu.au (B.M. Abbott).
http://dx.doi.org/10.1016/j.bmc.2014.06.018
0968-0896/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

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N. J. O’Brien et al. / Bioorg. Med. Chem. 22 (2014) 3879–3886
Scheme 1. Reagents and conditions: (a) For 5a and 5b: aniline or 4-methoxyaniline
(1.5 equiv), DIPEA, DMF, 100 °C, 16 h, 72–82%; (b) For 5c: 4-methoxyphenol,
Cs2CO3, DMF, rt, 2 h, 51%; (c) For 5d and 5e: phenyl- or 3-methoxyphenylboronic
acid (1.2 equiv), Na2CO3 (2 equiv), Pd(PPh3)4 (6 mol %), 4:1 1,4-dioxane/H2O, reflux,
2 h, 55–66%; (d) For 5f–5i: arylboronic acid (1.2 equiv), K3PO4 (2 equiv), Pd(OAc)2
(2 mol %), CyJohnPhos (4 mol %), 4:1 1,4-dioxane/H2O, reflux, 2 h, 41–47%; (e) For
5j: K2CO3 (3 equiv), 4:1 MeOH/H2O, reflux, 16 h, 82%.
moieties was achieved using the modified Suzuki conditions to
give compounds 5g–i, again with 15–20% of the bis(substituted)
side-products. These could be totally removed in the first two
cases; the bis(3,4,5-trimethoxyphenyl) substituted side-product 6
was successfully isolated and characterised, thus confirming the
identities of these side-products. This new catalyst system was
highly advantageous to the overall synthesis of these inhibitors
as the tosyl protecting group was no longer required. Finally, a
methoxy substituent was installed by refluxing compound 3 in
aqueous methanol and base, which also removed the tosyl group,
to give compound 5j in a yield of 82%.
The 2-anilino-4-substituted-N-tosylpyrrolopyrimidines 8a–e
were synthesised by Buchwald–Hartwig cross-coupling22 of
compounds 5a–e with aniline 7, previously synthesised in our
laboratory,17 in yields of 77–85%. Then, base promoted detosyla-
tion22 provided the first set of desired 2-anilino-4-substituted-7H-
pyrrolopyrimdines 9a–e in good yields (Table 1). Buchwald–Hartwig
Figure 2. Molecular model of our previously synthesised target compound 1 bound
to the PDK1 binding site illustrating access to the hinge region (Hinge), catalytic
residues (Cat.), ribose pocket (R) and entrance region (E0) from the designed
scaffold.
The screening results of the first-generation analogues illus-
trated that the 2-anilino moiety containing the pyrrolidine urea
side chain was important in maintaining activity against PDK1,
as indicated by the work of Feldman et al.,19 but the overall size
of the inhibitors was of concern.
Thus, investigation into changes at the 4-position to decrease
the size of the compounds was undertaken in an attempt to
improve inhibitory activity. Whilst this work was in progress,
Blanchard et al. reported a series of 2-anilino-6-aryl-9H-purine
derivatives that selectively inhibited PDK1.18 These inhibitors were
similar to our compound 1 (Fig. 1), which displayed 46 6%
inhibition at 10 lM, but they possessed smaller substituted aryl
groups at the 6-position of the purine scaffold. The standout
inhibitors of the series were the 4-methoxy, 3,4-dimethoxy and
3,4-methylenedioxy substituted aryl systems, with the most
potent inhibitor, the 4-methoxyphenylpurine 2 reported to possess
Table 1
Synthesis of 2-anilino-4-substituted-7H-pyrrolopyrimidine derivatives 9a–j by Buch-
wald–Hartwig cross-coupling of compounds 5a–e and 5f–j with aniline 7, followed by
detosylation of intermediates 8a–e
an IC50 of 0.15 l
M.18 It was postulated that the smaller functional-
ity and the methoxy substituent might have contributed to the
increase of inhibitory activity towards PDK1.
Herein we report the synthesis of a series of second-generation
analogues, with variation at the 4-position of the 7H-pyrrolopyr-
imidine scaffold, and their biological evaluation against PDK1.
2. Results and discussion
The greater reactivity of the 4-chloro group in dichloro com-
pound 3 allowed functionality to be selectively added to this posi-
tion through nucleophilic displacement and Suzuki cross-coupling
reactions (Scheme 1). Reaction with aniline and 4-methoxyaniline
gave 5a and 5b in yields of 82% and 72%, respectively. Installation
of a 4-methoxyphenoxy moiety to give 5c was undertaken by
Entry
Substrate
Product
Yield (%)a,b
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
5g
5h
5i
8a, 9a
8b, 9b
8c, 9c
8d, 9d
8e, 9e
9f
9g
9h
9i
9j
84, 87
81, 65
72, 68
85, 95
77, 65
78
68
70
55
65
reacting compound
3 with 4-methoxyphenol and caesium
carbonate. Suzuki cross-coupling, using standard conditions,20
gave the desired phenyl and 3-methoxyphenyl compounds 5d
and 5e in yields of 55% and 66%, respectively.
However, the same reaction with 4-methoxyphenylboronic acid
failed. Modified Suzuki cross-coupling conditions reported by
Dodonova et al.,21 using a more active catalyst system and the
unprotected 2,4-dichloropyrrolopyrimidine 4, provided the desired
5f with 15–20% of the bis(4-methoxyphenyl) substituted
side-product, which could only be partially removed by flash chro-
matography and recrystallisation. Installation of 3,4-dimethoxy-
phenyl, 3,4,5-trimethoxyphenyl and 3,4-(methylenedioxy)phenyl
10
5j
Reagents and conditions: (a) aniline 7 (1.2 equiv), K2CO3 (2.2 equiv), Pd2(dba)3
(5 mol %), XPhos (10 mol %), t-BuOH, reflux, 16 h; (b) K2CO3 (3 equiv), 4:1 MeOH/
H2O, reflux, 3 h.
Yields obtained for compounds 9a–e are from the Buchwald–Hartwig cross-
coupling and destosylation reactions, respectively.
a
b
Yields obtained for compounds 9f–j are from the Buchwald–Hartwig cross-
coupling reaction.

N. J. O’Brien et al. / Bioorg. Med. Chem. 22 (2014) 3879–3886
3881
cross-coupling of compounds 5f–j with aniline 7 gave direct transfor-
mation to the second set of 2-anilino-4-substituted-7H-pyrrolopy-
rimdines 9f–j in yields from 55–78%.
Second-generation analogues 9a–j were screened against the
isolated PDK1 enzyme at a concentration of 10 lM to give a
percentage inhibition (Table 2). As anticipated, removal of the
3-(pyridin-3-ylmethoxy) side chain, from target compound 1,
resulted in an overall increase of inhibitory activity against PDK1.
Truncation to a phenyl group (9d) gave an improved inhibition,
whilst introduction of an NH spacer (9a) decreased the inhibitory
activity. Installation of a 4-methoxyanilino group (9b) had the
same effect, suggesting that an N-linker in this position is not
100
50
0
Compound 9f
Compound 9j
Table 2
0.001
0.01
0.1
1
10
100
2-Anilino-4-substituted-7H-pyrrolopyrimidine derivatives 9a–j and their percentage
Concentration (µM)
inhibitory activity against PDK1
Figure 3. Dose–response curves of compounds 9f and 9j, where the IC50 SD is of
at least 3 independent experiments (radiometric assay). Curves were fitted using
GraphPad Prism software.
favourable. Inhibition was restored by
a 4-methoxyphenoxy
moiety (9c), showing that an O-linker is preferable and the
addition of a methoxy group had some improved inhibitory effect.
3-Methoxyphenyl (9e), 4-methoxyphenyl (9f), 3,4-dimethoxyphenyl
(9g) and 3,4-methylenedioxyphenyl (9i) groups all increased the
inhibitory activity, whilst the bulkier 3,4,5-trimethoxyphenyl
group (9h) decreased inhibition.
Overall, these results illustrate that the C–C linker bond was
preferred for kinase inhibition, and that a methoxy group on the
aryl ring has a positive effect. In particular, the methoxy group at
the 4-position is vital in providing improved hydrogen bond inter-
actions with the highly polar catalytic triangle-binding region.
Interestingly, compounds 9f, 9g and 9i provided an inhibitory
series comparable to their purine analogues.18 In a more radical
structural variation, the 4-methoxy analogue 9j was included in the
set and, unexpectedly, this compound showed significantly greater
activity than any of the preceding aryl substituted analogues.
Dose–response curves were completed for the two most potent
inhibitors of the series, compounds 9f and 9j, from which approx-
Compound
R1
% Inhibition at 10 l
Ma
1b
46
6
9a
9b
9c
9d
9e
46
45
65
59
48
2
7
3
2
6
imate IC50 values of 4.9 0.1 lM and 1.2 0.1 lM, respectively,
were determined (Fig. 3). Notably, the 4-methoxyphenyl
compound 9f was less active than the 4-methoxyphenylpurine 2
inhibitor reported by Blanchard et al.,18 suggesting that the extra
nitrogen of the purine scaffold may provide important physio-
chemical properties or interactions, such as a hydrogen bonding
role, within the ATP binding site.
3. Conclusion
9f
9g
9h
70
65
36
6
4
8
A series of second-generation 7H-pyrrolopyrimidine analogues
was produced to provide an insight into the effect of smaller func-
tionality on the catalytic residues of the PDK1 binding site. Biolog-
ical evaluation showed an overall increase in activity towards the
target kinase in comparison to the first-generation set. 4-Methoxy
compound 9j provides a potentially useful lead for the develop-
ment of new PDK1 inhibitors. Further investigation into the effect
of the extra nitrogen of the purine scaffold is needed for the two
most active compounds 9f and 9j and is currently underway.
4. Experimental
9i
9j
61
90
7
2
4.1. General methods
Melting points were recorded on a Reichert ‘Thermopan’ micro-
scope hot stage apparatus and are uncorrected. NMR spectra were
recorded on a Bruker AV-500 at 500.19 MHz for 1H nuclei and at
a
The mean SD of at least 3 independent experiments (radiometric assay).
b
17
Result from Ref.
.

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N. J. O’Brien et al. / Bioorg. Med. Chem. 22 (2014) 3879–3886
125.78 MHz for 13C nuclei at 300 K unless otherwise stated. All of
the chemical shifts were recorded as d values in parts per million
(ppm) and coupling constants (J) were recorded in Hertz (Hz) to
the nearest 0.5 Hz. The following abbreviations were used when
reporting 1H 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 Bru-
ker Daltronics Esquire 6000 ion trap mass spectrometer at 300 °C
with a scan rate of 5500 m/z/s, 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 electro-
spray were recorded on an Agilent 1290 Infinity. All data were
acquired and reference mass corrected via a dual-spray electro-
spray ionization (ESI) source, in positive mode, unless otherwise
stated. 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 (DavisilÒ LC60
used in the manuscript for synthesising compound 5a. The crude
product was recrystallised from toluene to give 5b (180 mg, 72%)
as a grey solid; mp 236–239 °C; dH (CDCl3): 8.06 (2H, d, J 8.0 Hz),
7.30 (2H, d, J 8.0 Hz), 7.24 (3H, d, J 9.0 Hz), 7.02 (1H, br s), 6.91
(2H, d, J 9.0 Hz), 5.51 (1H, br s), 3.83 (3H, s), 2.39 (3H, s); dC (CDCl3):
158.7, 156.7, 155.0, 152.1, 145.8, 134.5, 129.8, 129.7, 128.7, 127.5,
122.3, 114.6, 103.2, 103.1, 55.5, 21.7; m/z (ESI): 429.0 (M[35Cl]H+),
431.0 (M[37Cl]H+); HRMS (ESI): M[35Cl]H+, found 429.0789. C20H18-
ClN4O3S+ requires 429.0783.
4.2.3. 2-Chloro-4-(4-methoxyphenoxy)-7-tosyl-7H-pyrrolo[2,3-
d]pyrimidine (5c)
To a stirred solution of 4-methoxyphenol (87 mg, 0.70 mmol) in
DMF (10 mL) was added Cs2CO3 (229 mg, 0.70 mmol).
After 10 min, 2,4-dichloro-N-tosylpyrrolopyrimidine 3 (200 mg,
0.58 mmol) was added and the mixture was stirred at room
temperature for 30 min. The reaction mixture was diluted with
H2O (40 mL) and extracted with EtOAc (2 Â 30 mL). The combined
organic extracts were washed with brine (6 Â 25 mL), dried,
filtered and the solvent was removed in vacuo to give a grey residue.
The residue was purified by flash chromatography (20% EtOAc/
Hexanes) and recrystallised from n-PrOH to give 5c (150 mg, 51%)
as a white solid; mp 161–164 °C; dH (CDCl3): 8.10 (2H, d, J 8.0 Hz),
7.55 (1H, d, J 4.0 Hz), 7.32 (2H, d, J 8.0 Hz), 7.06 (2H, d, J 9.0 Hz),
6.90 (2H, d, J 9.0 Hz), 6.41 (1H, d, J 4.0 Hz), 3.81 (3H, s), 2.40 (3H,
s); dC (CDCl3): 163.0, 157.5, 154.1, 153.2, 146.2, 145.5, 134.3,
129.9, 128.7, 124.9, 122.2, 114.7, 106.6, 102.3, 55.6, 21.7; m/z
(ESI): 430.0 (M[35Cl]H+), 432.0 (M[37Cl]H+); HRMS (ESI): M[35Cl]H+,
found 430.0636. C20H17ClN3O4S+ requires 430.0623.
40–63 lm) as the stationary phase according to the method of Still
et al.23 All solvents used for flash chromatography, including tri-
ethylamine, 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: dichloro-
methane (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 sol-
vents and reagents was carried out by the procedures described
by Chai and Armarego.24 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 sulfate unless otherwise stated.
4.2.4. 2-Chloro-4-phenyl-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine
(5d)
To a solution of the 2,4-dichloro-N-tosylpyrrolopyrimidine 3
(1.00 g, 2.92 mmol) in 1,4-dioxane (40 mL) and H2O (10 mL) was
added phenylboronic acid (430 mg, 3.50 mmol) and Na2CO3
(620 mg, 5.84 mmol), and the mixture was degassed under argon.
To this mixture was added Pd(PPh3)4 (100 mg, 6 mol %) and the
reaction mixture was heated at reflux for 1 h, then cooled to room
temperature and the solvent was removed in vacuo. The residue
was dissolved in EtOAc (50 mL) and washed with H2O
(2 Â 30 mL), brine (2 Â 30 mL), dried, filtered and the solvent was
removed in vacuo to give a yellow residue. The residue was
purified by flash chromatography (10% EtOAc/Hexanes) and
recrystallised from EtOH to give 5d (600 mg, 55%) as a white solid;
mp 151–155 °C; dH (CDCl3): 8.14 (2H, d, J 8.0 Hz), 7.97 (2H, dd,
J 6.0, 2.0 Hz), 7.75 (1H, d, J 4.0 Hz), 7.52–7.51 (3H, m), 7.34 (2H,
d, J 8.0 Hz), 6.87 (1H, d, J 4.0 Hz), 2.41 (3H, s); dC (CDCl3): 160.6,
155.3, 152.9, 146.3, 136.0, 134.2, 131.1, 129.9, 129.0, 128.8,
127.1, 116.3, 104.1, 21.7; m/z (ESI): 384.0 (M[35Cl]H+), 386.0
(M[37Cl]H+); HRMS (ESI): M[35Cl]H+, found 384.0567. C19H15ClN3
O2S+ requires 384.0569.
4.2. Synthesis
4.2.1. 2-Chloro-N-phenyl-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (5a)
To
(200 mg, 0.58 mmol) in DMF (10 mL) was added DIPEA (204
1.17 mmol) and aniline (80
a
solution of 2,4-dichloro-N-tosylpyrrolopyrimidine
3
L,
l
lL, 0.88 mmol), and the reaction
mixture was heated to 100 °C overnight. The reaction mixture
was allowed to cool to room temperature, H2O (40 mL) was added
and the whole mixture was extracted with EtOAc (2 Â 30 mL). The
combined organic extracts were washed with brine (6 Â 25 mL),
dried, filtered and the solvent was removed in vacuo to give a dark
residue. The residue was purified by flash chromatography (40%
EtOAc/Hexanes) and recrystallised from EtOH to give 5a (0.19 g,
82%) as a light yellow solid; mp 195–199 °C; dH (DMSO-d6):
10.03 (1H, s), 7.98 (2H, d, J 8.0 Hz), 7.68 (1H, d, J 4.0 Hz), 7.66
(2H, d, J 8.0 Hz), 7.48 (2H, d, J 8.0 Hz), 7.39 (2H, t, J 8.0 Hz), 7.14
(1H, t, J 7.5 Hz), 7.01 (1H, br s), 2.38 (3H, s); dC (DMSO-d6): 154.6,
153.8, 150.5, 146.2, 138.3, 134.1, 130.2, 128.8, 127.7, 124.1,
123.4, 121.6, 104.6, 103.8, 21.1; m/z (ESI): 399.1 (M[35Cl]H+),
401.0 (M[37Cl]H+); HRMS (ESI): M[35Cl]H+, found 399.0674.
4.2.5. 2-Chloro-4-(3-methoxyphenyl)-7-tosyl-7H-pyrrolo[2,3-
d]pyrimidine (5e)
2,4-Dichloro-N-tosylpyrrolopyrimidine 3 (250 mg, 0.73 mmol)
was coupled with 3-methoxyphenylboronic acid according to the
procedure used in the manuscript for synthesising compound 5d.
Purification by flash chromatography (15% EtOAc/Hexanes) and
recrystallisation from EtOH afforded 5e (200 mg, 66%) as a white
solid; mp 142–146 °C; dH (CDCl3): 8.14 (2H, d, J 8.0 Hz), 7.74 (1H,
d, J 4.0 Hz), 7.53 (1H, t, J 8.0 Hz), 7.49 (1H, s), 7.41 (1H, t, J
8.0 Hz), 7.34 (2H, d, J 8.0 Hz), 7.05 (1H, dd, J 8.0, 2.5 Hz), 6.86
(1H, d, J 4.0 Hz), 3.86 (3H, s), 2.40 (3H, s); dC (CDCl3): 160.5,
160.1, 155.2, 152.9, 146.3, 137.4, 134.2, 130.0, 129.9, 128.8,
127.1, 121.4, 117.1, 116.4, 114.1, 104.2, 55.5, 21.7; m/z (ESI):
C
19H16 ClN4O2S+ requires 399.0677.
4.2.2. 2-Chloro-N-(4-methoxyphenyl)-7-tosyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (5b)
2,4-Dichloro-N-tosylpyrrolopyrimidine 3 (200 mg, 0.58 mmol)
was coupled with 4-methoxyaniline according to the procedure

N. J. O’Brien et al. / Bioorg. Med. Chem. 22 (2014) 3879–3886
3883
414.0 (M[35Cl]H+), 416.0 (M[37Cl]H+); HRMS (ESI): M[35Cl]H+,
found 414.0680. C20H17ClN3O3S+ requires 414.0674.
acid according to General Procedure A. Purification by flash
chromatography (20% EtOAc/Hexanes) and recrystallisation
from n-PrOH afforded an inseparable mixture (20:1) of 5i
and bis(3,4-methylenedioxyphenyl) substituted side-product
4.2.6. General Procedure A: Preparation of 2-chloropyrrolopyr-
imidines by monoligated palladium catalysed Suzuki–Miyaura
cross-coupling
(89 mg, 41%) as
a light yellow solid; mp 258–264 °C; dH
(DMSO-d6): 12.43 (1H, s), 7.76 (1H, dd, J 8.0, 2.0 Hz), 7.67
(1H, d, J 4.0 Hz), 7.64 (1H, d, J 2.0 Hz), 7.13 (1H, d, J 8.0 Hz),
4.2.6.1. 2-Chloro-4-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrim-
idine (5f). To
a
solution of 2,4-dichloropyrrolopyrimidine
4
6.93 (1H, d,
J 4.0 Hz), 6.16 (2H, s); m/z (ESI): 274.0
(200 mg, 1.06 mmol) in 1,4-dioxane (10 mL) and H2O (2.5 mL) was
added 4-methoxyphenylboronic acid (193 mg, 1.28 mmol) and
K3PO4 (542 mg, 2.55 mmol), and the mixture was degassed under
argon. To this mixture was added Pd(OAc)2 (12 mg, 2 mol %) and
CyJohnPhos (37 mg, 4 mol %) and the reaction mixture was heated
at reflux for 2 h, then cooled to room temperature and the solvent
was removed in vacuo. The residue was dissolved in EtOAc
(50 mL) and washed with H2O (2 Â 30 mL), brine (2 Â 30 mL), dried,
filtered and the solvent was removed in vacuo to give the crude
product. The crude product was purified by flash chromatography
(1% MeOH/CH2Cl2) and recrystallised from EtOH to give an insepara-
ble mixture (20:1) of 5f and bis(4-methoxyphenyl) substituted side-
product (130 mg, 47%) as a light yellow solid; mp 240–246 °C (lit.21
249–249.6 °C); dH (DMSO-d6): 12.41 (1H, s), 8.16 (2H, d, J 9.0 Hz),
7.66 (1H, dd, J 3.5, 2.5 Hz), 7.15 (2H, d, J 9.0 Hz), 6.94 (1H, dd, J
3.5, 2.5 Hz), 3.87 (3H, s); m/z (ESI): 260.0 (M[35Cl]H+), 262.0
(M[37Cl]H+).
(M[35Cl]H+), 276.0 (M[37Cl]H+).
4.2.6.5.
(5j).
2-Chloro-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine
To a solution of 2,4-dichloro-N-tosylpyrrolopyrimidine
3 (250 mg, 0.73 mmol) in MeOH (5 mL) and H2O (3 mL) was added
K2CO3 (303 mg, 2.19 mmol) and the mixture was heated at reflux
for 5 h. The reaction mixture was cooled, diluted out with H2O
(30 mL) and the mixture was extracted with EtOAc (2 Â 30 mL).
The combined organics extracts were washed with brine
(2 Â 25 mL), dried, filtered and the solvent was removed in vacuo
to give a yellow residue. The residue was recrystallised from
toluene to give the 5j (110 mg, 82%) as a white solid; mp
190–195 °C (dec.); dH (DMSO-d6): 12.23 (1H, s), 7.38 (1H, d,
J 3.5 Hz), 6.51 (1H, d, J 3.5 Hz), 4.04 (3H, s); dC (DMSO-d6): 162.8,
153.1, 150.6, 124.9, 103.2, 98.4, 54.2; m/z (ESI): 183.9
(M[35Cl]H+), 185.9 (M[37Cl]H+); HRMS (ESI): M[35Cl]H+, found
184.0275. C7H7ClN3O+ requires 184.0273.
4.2.7. General Procedure B: Preparation of 2-anilino-N-tosyl-
and 2-anilino-7H-pyrrolopyrimidines via Buchwald–Hartwig
cross-coupling
4.2.7.1. N-(3-((4-(Phenylamino)-7-tosyl-7H-pyrrolo[2,3-d]pyrim-
idin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide (8a).
4.2.6.2.
2-Chloro-4-(3,4-dimethoxyphenyl)-7H-pyrrolo[2,3-d]
pyrimidine (5g). 2,4-Dichloropyrrolopyrimidine 4 (150 mg,
0.80 mmol) was coupled with 3,4-dimethoxyphenylboronic
acid according to General Procedure A. Purification by flash
chromatography (30–40% EtOAc/Hexanes) and recrystallisation
A
mixture of 2-chloro-N-tosylpyrrolopyrimidine 5a (250 mg,
0.63 mmol), aniline 7 (154 mg, 0.75 mmol) and K2CO3 (134 mg,
9.69 mmol) in anhydrous t-BuOH (15 mL) was degassed under
argon. To this mixture was added XPhos (21 mg, 10 mol %) and Pd2
(dba)3 (20 mg, 5 mol %) and the reaction was refluxed overnight,
then allowed to cool. To this was added H2O (40 mL) and the mix-
ture was extracted with CH2Cl2 (3 Â 30 mL). The combined organic
extracts were washed with brine (2 Â 40 mL), dried, filtered and the
solvent was removed in vacuo to give a dark red residue. The residue
was purified by flash chromatography (50% EtOAc/Hexanes) and
recrystallised from toluene to give 8a (300 mg, 84%) as a beige solid;
mp 143–146 °C; dH (DMSO-d6): 9.34 (1H, s), 9.18 (1H, s), 8.01 (2H, d,
J 8.0 Hz), 7.93 (1H, s), 7.87 (2H, d, J 8.0 Hz), 7.74 (1H, d, J 1.5 Hz), 7.67
(1H, d, J 8.0 Hz), 7.38 (2H, d, J 8.0 Hz), 7.35 (1H, d, J 4.0 Hz), 7.30 (2H,
t, J 8.0 Hz), 7.17 (1H, t, J 8.0 Hz), 7.05 (1H, d, J 8.0 Hz), 7.02 (1H, t,
J 7.0 Hz), 6.97 (1H, d, J 4.0 Hz), 3.38–3.35 (4H, m), 2.33 (3H, s),
1.86–1.84 (4H, m); dC (DMSO-d6): 156.7, 154.02, 153.99, 152.2,
145.5, 140.7, 140.4, 139.7, 134.7, 129.9, 128.5, 127.9, 127.6, 122.5,
120.6, 119.4, 113.6, 113.4, 111.8, 104.3, 99.4, 45.6, 25.0, 21.1; m/z
(ESI): 568.2 (MH+); HRMS (ESI): MH+, found 568.2122. C30H30N7O3
S+ requires 568.2126.
from EtOH afforded 5g (105 mg, 45%) as
a yellow solid;
mp 227–230 °C; dH (CDCl3): 10.99 (1H, s), 7.76 (1H, dd, J 8.0,
2.0 Hz), 7.74 (1H, d, J 2.0 Hz), 7.42 (1H, dd, J 3.5, 2.5 Hz), 7.01
(1H, d, J 8.0 Hz), 6.86 (1H, dd, J 3.5, 2.0 Hz), 4.01 (3H, s), 3.97
(3H, s); dC (CDCl3): 159.4, 154.0, 153.1, 151.5, 149.3, 129.7,
126.2, 122.6, 113.9, 111.7, 110.9, 101.8, 56.12, 56.05; m/z
(ESI): 290.0 (M[35Cl]H+), 292.0 (M[37Cl]H+); HRMS (ESI):
M[35Cl]H+, found 290.0694. C14H13ClN3O+2 requires 290.0691.
4.2.6.3. 2-Chloro-4-(3,4,5-trimethoxyphenyl)-7H-pyrrolo[2,3-d]
pyrimidine (5h).
2,4-Dichloropyrrolopyrimidine 4 (100 mg,
0.53 mmol) was coupled with 3,4,5-trimethoxyphenylboronic
acid according to General Procedure A. Purification by flash
chromatography (30–40% EtOAc/Hexanes) and recrystallisation
from EtOH afforded 5h (70 mg, 41%) as a white solid; mp
232–235 °C; dH (CDCl3): 11.08 (1H, s), 7.44 (1H, dd, J 3.5,
2.0 Hz), 7.38 (2H, s), 6.85 (1H, dd, J 3.5, 2.0 Hz), 3.97 (6H, s),
3.93 (3H, s); dC (CDCl3): 159.5, 154.1, 153.6, 153.0, 140.7, 132.2,
126.6, 114.2, 106.5, 101.7, 61.0, 56.4; m/z (ESI): 320.1
(M[35Cl]H+), 322.1 (M[37Cl]H+); HRMS (ESI): M[35Cl]H+, found
320.0801. C15H15ClN3O+3 requires 320.0797.
4.2.7.2. N-(3-((4-((4-Methoxyphenyl)amino)-7-tosyl-7H-pyrrol-
o[2,3-d]pyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxam-
ide (8b). 2-Chloro-N-tosylpyrrolopyrimidine 5b (150 mg,
0.35 mmol) was coupled with aniline 7 according to General Proce-
dure B. Purification by flash chromatography (60% EtOAc/1% Et3N/
Hexanes) and recrystallisation from toluene afforded 8b (170 mg,
81%) as a tan solid; mp 138–142 °C; dH (CDCl3): 8.48 (1H, s), 7.96
(2H, d, J 8.5 Hz), 7.56 (1H, dd, J 8.0, 1.5 Hz), 7.32 (2H, d, J 9.0 Hz),
7.21 (1H, t, J 8.0 Hz), 7.18 (2H, d, J 8.0 Hz), 7.10 (1H, d, J 4.0 Hz),
6.89–6.86 (4H, m), 6.69 (1H, s), 6.45 (1H, s), 5.83 (1H, br s), 3.81
(3H, s), 3.44–3.41 (4H, m), 2.31 (3H, s), 1.92–1.89 (4H, m); dC
(CDCl3): 157.5, 156.4, 155.7, 154.0, 153.0, 145.3, 140.5, 140.2,
135.3, 130.9, 129.8, 129.2, 127.7, 126.0, 119.6, 114.3, 112.3,
112.1, 109.4, 103.6, 99.0, 55.5, 45.7, 25.6, 21.6; m/z (ESI): 598.2
Side-product
2,4-bis(3,4,5-trimethoxyphenyl)-7H-pyrrolo[2,
3-d]pyrimidine (6) was eluted from the flash chromatography
(60% EtOAc/Hexanes) and recrystallised from EtOH as a light yel-
low solid (22 mg); mp 186–189 °C; dH (DMSO-d6): 12.33 (1H, br
s), 7.90 (2H, s), 7.65 (1H, d, J 2.0 Hz), 7.60 (2H, s), 6.96 (1H, d,
J 3.5 Hz), 3.95 (6H, s), 3.92 (6H, s), 3.78 (3H, s), 3.75 (3H, s); dC
(DMSO-d6): 155.6, 154.9, 153.8, 153.1, 152.9, 139.3, 139.0, 133.9,
133.5, 128.0, 112.7, 105.7, 104.5, 100.3, 60.12, 60.08, 55.8, 55.7;
m/z (ESI): 452.2 (MH+); HRMS (ESI): MH+, found 452.1822.
C
24H26N3O+6 requires 452.1817.
4.2.6.4. 4-(Benzo[d][1,3]dioxol-5-yl)-2-chloro-7H-pyrrolo[2,3-d]
pyrimidine (5i). 2,4-Dichloropyrrolopyrimidine 4 (150 mg,
0.80 mmol) was coupled with 3,4-(methylenedioxy)phenylboronic

3884
N. J. O’Brien et al. / Bioorg. Med. Chem. 22 (2014) 3879–3886
(MH+); HRMS (ESI): MH+, found 598.2238. C31H32N7O4S+ requires
598.2231.
EtOAc/Hexanes to give the title compound 9a (105 mg, 87%) as a
white solid; mp 144–147 °C; dH (CDCl3): 9.86 (s, 1H), 7.99 (dd
app t, J 1.5 Hz, 1H), 7.58 (d, J 8.0 Hz, 1H), 7.33 (t, J 8.0 Hz, 1H),
7.15–7.06 (m, 5H), 6.90 (s, 1H), 8.54 (dd, J 3.5, 2.0 Hz, 1H), 6.18
(s, 1H), 6.05 (dd, J 3.5, 2.0 Hz, 1H), 3.32–3.29 (m, 4H), 1.86–1.83
(m, 4H); dC (CDCl3): 155.8, 154.4, 154.0, 152.9, 140.9, 139.9,
139.1, 129.1, 128.9, 123.7, 121.8, 119.2, 113.5, 112.8, 110.3, 98.6,
98.2, 45.7, 25.5; m/z (ESI): 414.2 (MH+); HRMS (ESI): MH+, found
414.2034. C23H24N7O+ requires 414.2037.
4.2.7.3. N-(3-((4-(4-Methoxyphenoxy)-7-tosyl-7H-pyrrolo[2,3-d]
pyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide
(8c). 2-Chloro-N-tosylpyrrolopyrimidine 5c (100 mg, 0.23 mmol)
was coupled with aniline 7 according to General Procedure B.
Purification by flash chromatography (60% EtOAc/Hexanes) and
recrystallisation from CCl4 afforded 8c (100 mg, 72%) as a tan solid;
mp 202–204 °C; dH (CDCl3): 8.25 (1H, br s), 7.98 (2H, d, J 8.5 Hz),
7.56 (1H, br d, J 8.0 Hz), 7.37 (1H, br s), 7.28 (1H, d, J 4.0 Hz),
7.20 (2H, d, J 8.0 Hz), 7.17 (1H, t, J 8.0 Hz), 7.07 (2H, d, J 9.0 Hz),
6.91 (2H, d, J 9.0 Hz), 6.87 (1H, dd, J 8.0, 1.5 Hz), 6.42 (1H, s),
6.31 (1H, d, J 4.0 Hz), 3.82 (3H, s), 3.45–3.42 (4H, m), 2.33 (3H, s),
1.93–1.91 (4H, m); dC (CDCl3): 162.9, 157.3, 155.3, 154.0, 145.8,
145.8, 140.2, 139.6, 134.9, 130.0, 129.3, 127.8, 122.6, 121.6,
114.6, 113.0, 112.5, 109.5, 103.5, 100.9, 55.6, 45.7, 25.6, 21.6; m/z
(ESI): 599.2 (MH)+; HRMS (ESI): MH+, found 599.2080. C31H31N6O5-
S+ requires 599.2072.
4.2.8.2. N-(3-((4-((4-Methoxyphenyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide
(9b). 2-Anilino-N-tosylpyrrolopyrimidine 8b (150 mg, 0.25 mmol)
was treated according to General Procedure C. Purification by flash
chromatograhy (80–100% EtOAc/Hexanes) afforded the title
compound 9b (73 mg, 65%) as a beige solid; mp 132–136 °C; dH
(CDCl3): 9.01 (1H, br s), 7.98 (1H, dd app t, J 2.0 Hz), 7.45 (2H, d,
J 9.0 Hz), 7.15 (1H, t, J 8.0 Hz), 7.10 (1H, ddd, J 8.0, 2.0, 1.0 Hz),
7.04 (1H, ddd, J 8.0, 2.0, 1.0 Hz), 6.90 (2H, d, J 9.0 Hz), 6.86 (1H,
s), 6.69 (1H, s), 6.63 (1H, dd, J 3.5, 2.0 Hz), 6.12 (1H, s), 5.88 (1H,
dd, 3.5, 2.0 Hz), 3.81 (3H, s), 3.39–3.37 (4H, m), 1.93–1.90 (4H,
m); dC (CDCl3): 156.8, 155.8, 155.3, 153.9, 153.0, 141.0, 139.9,
131.9, 129.1, 125.1, 118.7, 114.1, 113.2, 112.5, 110.0, 99.2, 97.6,
55.5, 45.7, 25.5; m/z (ESI): 444.2 (MH+); HRMS (ESI): MH+, found
444.2148. C24H26N7O+2 requires 444.2143.
4.2.7.4. N-(3-((4-Phenyl-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)phenyl)pyrrolidine-1-carboxamide (8d). 2-Chloro-N-
tosylpyrrolopyrimidine 5d (200 mg, 0.52 mmol) was coupled with
aniline 7 according to General Procedure B. Purification by flash
chromatography (60% EtOAc/Hexanes) and recrystallisation from
CCl4 afforded 8d (245 mg, 85%) as
a yellow solid; mp
126–129 °C; dH (DMSO-d6): d 9.74 (1H, s), 8.08–8.04 (5H, m),
7.92 (1H, br s), 7.68 (1H, d, J 9.0 Hz), 7.66 (1H, d, J 4.0 Hz), 7.56
(3H, m), 7.39 (2H, d, J 8.5 Hz), 7.24 (1H, t, J 8.5 Hz), 7.10 (1H, d,
J 8.0 Hz), 6.98 (1H, d, J 4.0 Hz), 3.40–3.38 (4H, m), 2.33 (3H, s),
1.87–1.84 (4H, m); dC (DMSO-d6): 158.8, 156.7, 154.0, 153.5,
146.0, 140.6, 140.5, 136.8, 134.2, 130.6, 130.0, 128.9, 128.7,
128.1, 127.9, 123.7, 113.9, 113.1, 111.6, 109.7, 105.3, 45.7, 25.1,
21.1; m/z (ESI): 553.2 (MH+); HRMS (ESI): MH+, found 553.2012.
4.2.8.3. N-(3-((4-(4-Methoxyphenoxy)-7H-pyrrolo[2,3-d]pyrimi-
din-2-yl)amino)phenyl)pyrrolidine-1-carboxamide (9c).
2-Anilino-N-tosylpyrrolopyrimidine 8c (80 mg, 0.13 mmol) was
treated according to General Procedure C. Purification by flash
chromatography (90–100% EtOAc/Hexanes) and recrystallisation
from aqueous EtOH afforded the title compound 9c (41 mg, 68%)
as a white solid; mp 200–203 °C; dH (DMSO-d6): 11.48 (1H, br s),
8.91 (1H, s), 7.89 (1H, s), 7.55 (1H, dd app t, J 2.0 Hz), 7.34–7.32
(1H, m), 7.21 (2H, d, J 9.0 Hz), 7.02 (1H, dd, J 3.5, 2.0 Hz), 6.99
(2H, d, J 9.0 Hz), 6.95–6.91 (2H, m), 6.12 (1H, dd, J 3.5, 2.0 Hz),
3.79 (3H, s), 3.37–3.34 (4H, m), 1.86–1.83 (4H, m); dC (CDCl3):
163.0, 156.9, 155.3, 155.1, 153.9, 146.6, 140.6, 139.7, 129.0,
122.9, 120.3, 114.4, 113.0, 112.7, 109.7, 99.6, 99.1, 55.6, 45.8,
25.6; m/z (ESI): 445.2 (MH+); HRMS (ESI): MH+, found 445.1988.
C24H25N6O+3 requires 445.1983.
C
30H29N6O3S+ requires 553.2017.
4.2.7.5. N-(3-((4-(3-Methoxyphenyl)-7-tosyl-7H-pyrrolo[2,3-
d]pyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide
(8e). 2-Chloro-N-tosylpyrrolopyrimidine 5e (130 mg, 0.31 mmol)
was coupled with aniline 7 according to General Procedure B.
Purification by flash chromatography (60% EtOAc/Hexanes) and
recrystallisation from toluene afforded 8e (141 mg, 77%) as a tan
solid; mp 111–115 °C; dH (CDCl3): 8.54 (1H, t, J 1.5 Hz), 8.00 (2H,
d, J 8.5 Hz), 7.65 (1H, dd, J 8.0, 1.5 Hz), 7.49–7.46 (2H, m), 7.44
(1H, d, J 4.0 Hz), 7.40 (1H, t, J 8.0 Hz), 7.31 (1H, s), 7.28 (1H, t, J
8.0 Hz), 7.20 (2H, d, J 8.0 Hz), 7.03 (1H, ddd, J 8.0, 2.5, 1.0 Hz),
6.98 (1H, dd, J 8.0, 1.5 Hz), 6.73 (1H, d, J 4.0 Hz), 6.54 (1H, s),
3.86 (3H, s), 3.47–3.45 (4H, m), 2.32 (3H, s), 1.94–1.91 (4H, m);
dC (CDCl3): 160.0, 159.8, 156.4, 154.0, 153.5, 145.7, 140.4, 140.2,
138.7, 135.0, 130.0, 129.8, 129.4, 127.8, 123.3, 121.0, 116.4,
113.6, 112.6, 112.4, 110.9, 109.2, 105.4, 55.4, 45.7, 25.7, 21.6; m/z
(ESI): 583.2 (MH+); HRMS (ESI): MH+, found 583.2129. C31H31N6O4-
S+ requires 583.2122.
4.2.8.4.
N-(3-((4-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)
2-Anilino-N-
amino)phenyl)pyrrolidine-1-carboxamide (9d).
tosylpyrrolopyrimidine 8d (170 mg, 0.30 mmol) was treated
according to General Procedure C. Purification by flash chromatog-
raphy (70% EtOAc/Hexanes) and recrystallisation from EtOAc/
Hexanes afforded the title compound 9d (116 mg, 95%) as a yellow
solid; mp 128–132 °C; dH (CDCl3): 10.32 (1H, s), 8.15 (1H, dd app t,
J 2.0 Hz), 8.08 (2H, dd, J 8.0, 1.5 Hz), 7.53–7.48 (3H, m), 7.39 (1H, s),
7.20 (1H, t, J 8.0 Hz), 7.17 (1H, dt, J 7.5, 2.0 Hz), 7.04 (1H, dt, J 7.5,
2.0 Hz), 6.67 (1H, dd, J 3.5, 2.0 Hz), 6.56 (1H, dd, J 3.5, 2.0 Hz), 6.32
(1H, s), 3.31–3.29 (4H, m), 1.83–1.81 (4H, m); dC (CDCl3): 158.6,
156.0, 154.1, 153.9, 140.8, 140.2, 138.3, 129.9, 129.2,
128.7, 128.6, 123.1, 113.3, 112.8, 110.1, 109.8, 101.1, 45.7, 25.5;
m/z (ESI): 399.2 (MH+); HRMS (ESI): MH+, found 399.1926.
4.2.8. General Procedure C: Preparation of 2-anilino-7H-pyrrol-
opyrimidine derivatives via base promoted detosylation
4.2.8.1. N-(3-((4-(Phenylamino)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)phenyl)pyrrolidine-1-carboxamide (9a). To a solution
of 2-anilino-N-tosylpyrrolopyrimidine 8a (165 mg, 0.29 mmol) in
MeOH (5 mL) and H2O (3 mL) was added K2CO3 (165 mg,
1.20 mmol) and the mixture was refluxed for 3 h. To the cooled
solution was added H2O (30 mL) and the whole mixture was
extracted with EtOAc (2 Â 30 mL). The combined organic extracts
were washed with brine (2 Â 30 mL), dried, filtered and the solvent
was removed in vacuo to give a yellow residue. The residue was
purified by flash chromatography (EtOAc) and recrystallised from
C
23H23N6O+ requires 399.1928.
4.2.8.5. N-(3-((4-(3-Methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimi-
din-2-yl)amino)phenyl)pyrrolidine-1-carboxamide (9e). 2-Ani-
lino-N-tosylpyrrolopyrimidine 8e (100 mg, 0.17 mmol) was
treated according to General Procedure C. Purification by flash
chromatography (80–90% EtOAc/Hexanes) and recrystallisation
from n-PrOH afforded the title compound 9e (48 mg, 65%) as a
brown solid; mp 115–119 °C; dH (DMSO-d6): 11.60 (1H, s), 9.15
(1H, s), 7.98 (1H, s), 7.88 (1H, dd app t, J 2.0 Hz), 7.76 (1H, ddd, J

N. J. O’Brien et al. / Bioorg. Med. Chem. 22 (2014) 3879–3886
3885
8.0, 2.0, 1.0 Hz), 7.70 (1H, dd, J 2.5, 1.5 Hz), 7.53 (1H, ddd, J 8.0, 2.0,
1.0 Hz), 7.48 (1H, t, J 8.0 Hz), 7.26 (1H, dd, J 3.5, 2.0 Hz), 7.11 (1H, t,
J 8.0 Hz), 7.09 (1H, ddd, J 8.0, 2.0, 1.0 Hz), 7.01 (1H, ddd, J 8.0, 2.0,
1.0 Hz), 6.67 (1H, dd, J 3.5, 2.0 Hz), 3.86 (3H, s), 3.39–3.36 (4H, m),
1.87–1.85 (4H, m); dC (DMSO-d6): 159.4, 156.2, 156.0, 154.4, 154.0,
141.4, 140.5, 139.7, 129.7, 127.9, 124.3, 121.0, 115.9, 113.3, 113.2,
112.7, 111.1, 108.8, 100.2, 55.1, 45.7, 25.0; m/z (ESI): 429.2 (MH+);
HRMS (ESI): MH+, found 429.2038. C24H25N6O+2 requires 429.2034.
2.0 Hz), 7.74 (1H, d, J 1.5 Hz), 7.48 (1H, ddd, J 8.0, 2.0, 1.0 Hz),
7.24 (1H, dd, J 3.5, 2.0 Hz), 7.10 (1H, t, J 8.0 Hz), 7.09 (1H, d, J
8.0 Hz), 7.00 (1H, ddd, J 8.0, 2.0, 1.0 Hz), 6.67 (1H, dd, J 3.5,
1.5 Hz), 6.13 (2H, s), 3.40–3.37 (4H, m), 1.87–1.85 (4H, m); dC
(DMSO-d6): 155.79, 155.77, 148.7, 147.7, 141.4, 140.5, 132.4,
127.8, 124.0, 123.2, 112.9, 112.5, 110.8, 108.29, 108.26, 108.19,
101.4, 100.2, 45.6, 25.0; m/z (ESI): 443.2 (MH+); HRMS (ESI):
MH+, found 443.1833. C24H23N6O+3 requires 443.1827.
4.2.8.6. N-(3-((4-(4-Methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimi-
din-2-yl)amino)phenyl)pyrrolidine-1-carboxamide (9f). 2-Chlo-
ropyrrolopyrimidine 5f (75 mg, 0.29 mmol) was coupled with
aniline 7 according to General Procedure B. Purification by flash
chromatography (1–2% MeOH/CH2Cl2) afforded the title compound
9f (88 mg, 78%) as a brown solid; mp 118–122 °C; dH (CDCl3): 9.06
(1H, s), 8.09 (2H, d, J 9.0 Hz), 8.03 (1H, dd app t, J 2.0 Hz), 7.30–7.28
(1H, m), 7.21 (1H, t, J 8.0 Hz), 7.16 (1H, br s), 7.04 (2H, d, J 9.0 Hz),
7.03 (1H, ddd, J 8.0, 2.0, 1.0 Hz), 6.92 (1H, dd, J 3.5, 2.0 Hz), 6.65
(1H, dd, J 3.5, 2.0 Hz), 6.20 (1H, s), 3.88 (3H, s), 3.43–3.40 (4H,
m), 1.94–1.91 (4H, m); dC (CDCl3): 161.1, 158.2, 153.9, 154.0,
153.9, 141.0, 140.0, 130.9, 130.2, 129.2, 122.5, 114.0, 113.1,
112.5, 109.8, 109.2, 101.4, 55.4, 45.7, 25.5; m/z (ESI): 429.2
(MH+); HRMS (ESI): MH+, found 429.2040. C24H25N6O+2 requires
429.2034.
4.2.8.10.
N-(3-((4-Methoxy-7H-pyrrolo[2,3-d]pyrimidin-2-yl)
2-Chloropyr-
amino)phenyl)pyrrolidine-1-carboxamide (9j).
rolopyrimidine 5j (78 mg, 0.42 mmol) was coupled with aniline 7
according to General Procedure B. Purification by flash chromatog-
raphy (80% EtOAc/Hexanes) and recrystallisation from toluene
afforded the title compound 9j (97 mg, 65%) as a grey solid; mp
200–202 °C; dH (DMSO-d6): 11.38 (1H, s), 8.99 (1H, s), 7.97 (1H,
s), 7.88 (1H, dd app t, J 1.5 Hz), 7.43 (1H, d, J 8.0 Hz), 7.09 (1H, t,
J 8.0 Hz), 6.97–6.96 (2H, m), 6.29 (1H, dd, J 3.5, 1.5 Hz), 4.03 (3H,
s), 3.38–3.35 (4H, m), 1.86–1.84 (4H, m); dC (DMSO-d6): 162.5,
155.3, 154.0, 141.3, 140.5, 127.8, 120.6, 113.0, 112.6, 110.9, 98.2,
98.1, 55.1, 45.7, 25.0; m/z (ESI): 353.2 (MH+); HRMS (ESI): MH+,
found 353.1724. C18H21N6O+2 requires 353.1721.
4.3. Biological screening
4.2.8.7. N-(3-((4-(3,4-Dimethoxyphenyl)-7H-pyrrolo[2,3-d]pyrimi-
All compounds were assayed using a radiometric in vitro kinase
assay that was in accordance of Biondi et al.,25 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).1 PDK1
enzyme was purchased from Invitrogen at a purity of 70% and
din-2-yl)amino)phenyl)pyrrolidine-1-carboxamide
(9g).
2-Chloropyrrolopyrimidine 5g (65 mg, 0.22 mmol) was coupled with
aniline 7 according to General Procedure B. Purification by flash chro-
matography (90–100% EtOAc/Hexanes) and recrystallisation from
aqueous n-PrOH afforded the title compound 9g (69 mg, 68%) as a
yellow solid; mp 146–150 °C; dH (DMSO-d6): 11.53 (1H, s), 9.07
(1H, s), 7.98 (1H, s), 7.88 (1H, dd app t, J 2.0 Hz), 7.81–7.80 (2H, m),
7.54 (1H, ddd, J 8.0, 2.0, 1.0 Hz), 7.24–7.23 (1H, m), 7.14 (1H, d, J
8.0 Hz), 7.12 (1H, t, J 8.0 Hz), 6.99 (1H, ddd, J 8.0, 2.0, 1.0 Hz), 6.71
(1H, dd, J 3.5, 2.0 Hz), 3.88 (3H, s), 3.85 (3H, s), 3.39–3.36 (4H, m),
1.87–1.85 (4H, m); dC (DMSO-d6): 156.0, 155.8, 154.3, 154.0, 150.4,
148.7, 141.5, 140.5, 130.8, 127.8, 123.9, 121.7, 113.1, 112.7, 111.44,
111.43, 111.1, 108.3, 100.4, 55.6, 55.3, 45.7, 25.0; m/z (ESI): 459.2
(MH+); HRMS (ESI): MH+, found 459.2146. C25H27N6O+3 requires
459.2140.
diluted to a concentration of 6.25 ng/lL using 50 mM Tris buffer
(adjusted to pH 7.5) containing 0.1% b-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
À5 °C. [
c-
32P]-ATP was purchased from Perkin Elmer Easytides at
a 10 mCi/mL concentration and was stored at 2–8 °C. 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 fur-
ther purification.
4.2.8.8. N-(3-((4-(3,4,5-Trimethoxyphenyl)-7H-pyrrolo[2,3-d]
pyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide (9h)
2-Chloropyrrolopyrimidine 5h (45 mg, 0.14 mmol) was coupled
with aniline 7 according to General Procedure B. Purification by
flash chromatography (80–90% EtOAc/1% Et3N/Hexanes) and
recrystallisation from n-PrOH afforded the title compound 9h
(45 mg, 70%) as a white solid; mp 140–143 °C; dH (CDCl3): 9.77
(1H, s), 8.13 (1H, t, J 1.5 Hz), 7.36 (2H, s), 7.31 (1H, s), 7.28 (1H,
dd, J 8.0, 1.5 Hz), 7.20 (1H, t, J 8.0 Hz), 6.96 (1H, dd, J 8.0, 1.5 Hz),
6.84 (1H, dd, J 8.5, 2.0 Hz), 6.60 (1H, dd, J 8.5, 2.0 Hz), 3.95 (6H,
s), 3.92 (3H, s), 3.39–3.36 (4H, m), 1.90–1.88 (4H, m); dC (CDCl3):
158.2, 156.0, 154.3, 153.9, 153.4, 140.9, 140.0, 139.7, 133.7,
129.1, 122.8, 113.2, 112.7, 109.9, 109.3, 106.0, 101.3, 61.0, 56.3,
45.8, 25.6; m/z (ESI): 489.2 (MH+). HRMS (ESI): MH+, found
489.2250. C26H29N6O+4 requires 489.2245.
Acknowledgments
The authors would like to thank Dr. Suzanne 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.
Supplementary data
4.2.8.9. N-(3-((4-(Benzo[d][1,3]dioxol-5-yl)-7H-pyrrolo[2,3-d]
pyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide (9i)
2-Chloropyrrolopyrimidine 5i (40 mg, 0.15 mmol) was coupled
with aniline 7 according to General Procedure B. Purification by
flash chromatography (1–2% MeOH/CH2Cl2) and recrystallisation
from n-PrOH afforded the title compound 9i (35 mg, 55%) as a yel-
low solid; mp 234–238 °C; dH (DMSO-d6): 11.57 (1H, s), 9.11 (1H,
s), 8.00 (1H, s), 7.94 (1H, dd app t, J 2.0 Hz), 7.77 (1H, dd, J 8.0,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.06.018.
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