Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2012.02.107

A high-throughput screen against PknB, an essential serine-threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained.

Full text of DOI:10.1016/j.bmcl.2012.02.107

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3349–3353
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted aminopyrimidine protein kinase B (PknB) inhibitors
show activity against Mycobacterium tuberculosis
Timothy M. Chapman a, , Nathalie Bouloc a, Roger S. Buxton b, Jasveen Chugh a, Kathryn E.A. Lougheed b,
⇑
Simon A. Osborne a, Barbara Saxty a, Stephen J. Smerdon c, Debra L. Taylor a, David Whalley a
a Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, UK
b Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
c Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobac-
terium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which
was used as a starting point for SAR investigations. Although a significant improvement in enzyme affin-
ity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. How-
ever, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain
afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory con-
centrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed
equipotent compounds with improved selectivity against a human kinase panel to be obtained.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 2 February 2012
Revised 24 February 2012
Accepted 25 February 2012
Available online 14 March 2012
Keywords:
Protein kinase B
Mycobacterium tuberculosis
Aminopyrimidine
Structure–activity relationship
More than 14 million people worldwide suffer from tuberculo-
sis (TB) which in 2009 resulted in an estimated 1.7 million deaths.1
In particular, the increasing prevalence of multi-drug resistant
strains means that novel therapeutic approaches are urgently
needed. PknB is one of 11 serine–threonine protein kinases (STPKs)
found in Mycobacterium tuberculosis (M. tuberculosis) and has been
shown to be essential for bacterial growth,2 and therefore inhibi-
tion of PknB represents a potential therapeutic approach for the
treatment of TB. PknB is an attractive target as the kinase domain
contains less than 30% similarity with eukaryotic kinases, which
offers promise for achieving selectivity for the mycobacterial ki-
nase over those of the human host. M. tuberculosis is unusual
amongst bacteria in having a higher number of STPKs in compari-
son to the more common two-component signalling systems,3 and
of the other STPKs present, PknA has also been found to be essen-
tial while PknG has been reported to play a crucial role in the sur-
vival of mycobacteria within macrophages.4 Previous studies have
focused on the potential of PknB and PknG as drug targets in M.
tuberculosis, although the majority have reported significantly
lower activity against whole cells than against the purified
enzymes.2,4,5 This difference may be due in part to the nature of
the TB cell wall, which is recognised as difficult to permeate,
particularly due to its unique mycolic acid layer.6
A high throughput screen of our compound collection against
the isolated PknB enzyme was performed through measuring the
effect on the in vitro phosphorylation of the substrate GarA,7 and
this identified compound 1 (Fig. 1) as a 1.35 lM inhibitor of PknB,
which formed the starting point for our chemistry programme.
Initial SAR exploration around 1 was performed via synthetic
access from 2,4-dichloroquinazoline 3 (Scheme 1). Introduction
of the aminopyrazole was performed by nucleophilic substitution
at the 4-position, followed by either a Suzuki coupling to introduce
an aryl group or a second nucleophilic substitution to introduce a
second amino substituent at the 2-position.
Replacement of the pyrazole methyl substituent with cyclopro-
pyl gave a fourfold improvement in binding affinity (Table 1, 5a).
The 4-pyridyl group could also be substituted by other aryl groups
such as 4-fluorophenyl or phenyl while maintaining comparable
potency (5b–5e). Alkyl amines were also tolerated at this position,
H
N
N
HN
N
N
N
1
⇑
Corresponding author. Tel.: +44 20 8906 7100; fax: +44 20 8906 7200.
E-mail address: tim.chapman@tech.mrc.ac.uk (T.M. Chapman).
PknB IC50 = 1.35 µM
Figure 1. HTS hit 1.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.02.107

3350
T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3349–3353
H
H
N
N
N
N
N
N
R1
R1
O
Cl
N
HN
N
HN
N
a
b
c
NH
N
N
H
O
R2
or NHR2
5f-g
Cl
Cl
2
3
4
5a-e
Scheme 1. Reagents and conditions: (a) POCl3, N,N-dimethylaniline, reflux; (b) 3-amino-5-R1-pyrazole, EtOH, 20 °C; (c) R2B(OH)2, Pd(PPh3)2Cl2, Et3N, MeOH, microwave,
140 °C or R2NH2, EtOH, microwave, 150 °C.
Table 1
H
N
H
N
SAR of disubstituted quinazolines
N
N
N
N
Compound
R1
R2 or NHR2
PknB IC50
0.322
/l
Ma
Cl
HN
HN
N
a
b
N
5a
N
R1
or NHR1
N
6
Cl
N
7
Cl
H
5b
5c
0.453
1.162
8a-g 8h-n
F
CH3
Scheme 2. Reagents and conditions: (a) 3-Amino-5-cyclopropylpyrazole, DIPEA, i-
PrOH, 50 °C; (b) R1B(OH)2, Pd(PPh3)2Cl2, Et3N, MeOH, microwave, 150 °C or R1NH2,
DIPEA, i-PrOH, microwave, 170 °C.
F
F
5d
5e
0.223
0.440
concentrations (MICs) falling in the 63–250
active example was the 3-sulfonamidophenyl variant 8c at 31
l
M range. The most
lM.
Further improvements to the physical properties of the com-
pounds were sought, and a basic amine side chain was introduced
to give compounds with a lower logD. The 6-position was chosen
as a suitable point for appending this side-chain as modelling stud-
ies suggested that this region of the molecule was solvent exposed,
so would be amenable to the introduction of this group. A range of
basic side chains were introduced into this position, initially with a
phenyl group fixed at the 2-position (Scheme 3). The products,
shown in Table 3 demonstrated sub-100 nM enzyme potency and
generally improved MIC values, and compound 11e containing
the (R)-3-dimethylaminopyrrolidine basic side-chain displayed
5f
0.107
0.428
N
H
N
H
5g
a
Assay conditions are described in Ref. 8.
with cycloalkyl groups such as the cyclohexyl derivative 5f giving
the best IC50. However, the introduction of highly polar or charged
groups was not well tolerated at this position and led to a signifi-
cant loss in potency. Screening of these compounds against M.
tuberculosis in an Alamar blue cellular assay8 revealed that they
possessed weak or no cellular efficacy. Measured logD values for
these compounds were typically in excess of 4, and measurement
of their plasma–protein binding (ppb) revealed that they were
highly bound (>99%), leaving only a small free fraction available
to bind to the enzyme. Furthermore, the physical properties of
these compounds were not considered to be well disposed for
crossing the TB cell wall, as its mycolic acid layer contains barriers
to the passage of hydrophobic as well as hydrophilic molecules.6
In order to improve the physical properties of the compounds
with the aim of improving their cellular activity, it was decided
to reduce the logD of the compounds and explore the effect of this
in advancing the SAR. The quinazoline core was switched to a
pyrimidine, and while retaining the cyclopropylaminopyrazole at
the 4-position, a range of substituents including aryl and amino
groups were introduced at the 2-position to allow exploration of
the SAR (Scheme 2). This afforded compounds with improved po-
tency against the enzyme (Table 2), and amongst the aryl variants,
the 3-sulfonylphenyl compound 8d and 3-cyanophenyl derivative
8f showed the highest affinities at 86 and 87 nM, respectively.
For the amino compounds, in addition to the cyclohexyl and cyclo-
pentyl examples 8h and 8n at 84 and 115 nM, the substituted
phenylethylamines 8k and 8l showed promising enzyme affinities
of 74 and 64 nM, respectively. These compounds also demon-
strated improved efficacy against M. tuberculosis although this
was still relatively weak, with the majority of minimum inhibitory
an IC50 of 53 nM against the enzyme and a 16 lM MIC against M.
tuberculosis. Oxygen-linked basic side chains were also well toler-
ated, for example compound 11f, which showed an IC50 of 56 nM
against the enzyme and 31 lM MIC against the Mycobacterium.
Considering the enhanced enzyme binding affinity that had
resulted from the presence of a 3-cyano or 3-sulfonyl group on
the phenyl ring in the disubstituted variants (Table 2), these
substituents were introduced into the trisubstituted pyrimidines
aiming for a corresponding increase in enzyme potency and activ-
ity against M. tuberculosis. Starting with 4,6-dichloro-2-(methyl-
thio)pyrimidine 12, the cyclopropylaminopyrazole group and the
basic amine side-chain were sequentially introduced under nucle-
ophilic displacement conditions. This was followed by a Liebes-
kind–Srogl coupling9 under microwave heating on the S-methyl
pyrimidine 14 to install the desired aryl group (Scheme 4). How-
ever, the resultant compounds displayed modest gains in enzyme
affinity (approximately twofold for 15b and 15c) and no significant
improvement in activity against M. tuberculosis (Table 4), which re-
mained between 31 and 63 lM.
Despite the general improvement in MIC values obtained
through introduction of the basic side chain to the pyrimidine core,
efficacy against M. tuberculosis remained weaker than was desired.
In an effort to improve the cellular activity, it was decided to re-
place the aminopyrazole head group with an aminopyridine group.
This allowed the hydrogen bond donor count to be reduced, which
was considered desirable for improving permeability through the
M. tuberculosis cell wall. It also offered the potential for improved
kinase selectivity by reducing the reliance on the binding energy

T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3349–3353
3351
Table 2
SAR of disubstituted pyrimidines
Compound
R1 or NHR1
PknB IC50
/
l
Ma
M. tuberculosis MIC/l
Ma
8a
0.375
>500
F
O
8b
8c
8d
0.384
0.393
0.086
63
31
63
NH2
S
H
N
O
O
O
O
S
H
N
8e
0.241
63
S
O
N
O
8f
0.087
0.202
125
125
8g
N
8h
0.084
63
N
H
8i
8j
0.354
0.715
63
N
H
N
H
125
8k
8l
0.074
0.064
63
N
H
F
125
N
H
Cl
N
H
8m
8n
0.217
0.115
250
63
N
H
a
Assay conditions are described in Ref. 8.
H
H
N
using a palladium-catalysed amination for introduction of the ami-
nopyridine instead of nucleophilic displacement. Initial variants
showed a significant loss in binding affinity, but further explora-
tion of the SAR revealed that pyridines carrying a substituent at
the 4-position were optimal for enzyme inhibition and this al-
lowed the IC50 to be restored to the double-digit nanomolar range
(Table 5). However, in common with the aminopyrazoles, MIC val-
ues against M. tuberculosis remained in the micromolar region.
Compound 16c displayed the most promising activity with a MIC
N
N
N
Cl
N
HN
HN
N
a
b
N
N
N
Cl
Ph
R1R2N
or R1O
Ph
11a-e, 11f
Cl
N
Ph
9
10
Scheme 3. Reagents and conditions: (a) 3-Amino-5-cyclopropylpyrazole, DIPEA,
NaI, DMA, 100 °C; (b) R1R2NH, n-BuOH, microwave, 150–170 °C or R1OH, t-BuOK,
THF, microwave, 160 °C.
of 8 lM.
The selectivity of the inhibitors was examined against other M.
tuberculosis kinases and this revealed that they typically showed
cross-affinity with PknF, although they displayed only modest inhi-
bition of PknG. Aminopyrimidines featuring appended pyrazole
groups have been reported in the literature as inhibitors of a
gained at the kinase hinge region. These compounds were prepared
following a similar sequence to that described in Scheme 4, replac-
ing the aminopyrazole with the appropriate aminopyridine but

3352
T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3349–3353
Table 3
Table 5
SAR of trisubstituted pyrimidines
SAR of trisubstituted pyrimidines containing pyridine head group
Compound NR1R2 or OR1
PknB IC50
l
/
M. tuberculosis
m
Ma
MIC/
125
63
l
Ma
LogDb
N
R1
HN
N
N
11a
0.088
0.121
0.055
3.7
3.6
2.3
N
H
N
R2
11b
N
N
N
N
N
11c
11d
31
31
N
H
16a-d
H
0.073
0.053
2.3
3.8
N
N
N
N
Compound
R1
R2
PknB IC50
/
l
Ma
M. tuberculosis MIC/l
Ma
11e
11f
16
31
16a
16b
16c
16d
H
OMe
Cl
CN
H
H
1.99
1.13
0.176
0.040
31
31
8
0.056
3.4
N
CF3
CN
16
O
a
a
Assay conditions are described in Ref. 10.
Assay conditions are described in Ref. 8.
LogD values were measured by BioFocus.
b
aminopyridine head group variants using the PknB crystal struc-
ture (ref: 2FUM)12 from the PDB, and the predicted binding modes
of compounds 15c and 16d are shown in Figure 3. It can be seen
that the aminopyrazoles can form up to 3 hydrogen bonds with
the hinge region of the kinase, and the appended cyclopropyl group
can form a face-on lipophilic interaction with the methionine gate-
keeper residue. The cyano group on the phenyl ring points in the
direction of a lysine residue. By contrast, in the aminopyridine vari-
ants two hydrogen bonds with the hinge region can be formed, and
the 4-substituent on the pyridine head group is orientated in the
direction of the gatekeeper.
In summary, the starting point HTS hit was optimised for po-
tency against PknB, and initial modifications showed promising
gains in enzyme binding affinity but little or no cellular activity
against M. tuberculosis. Switching from the quinazoline to a pyrim-
idine core and introduction of a basic side chain improved the
physical properties of the molecules and resulted in improved
cellular activities, in the double-digit micromolar region. While
further optimisation gave improvements in enzyme binding affin-
ity, this did not translate into improved efficacy against M. tubercu-
losis. Replacement of the aminopyrazole head group with an
aminopyridine led to an initial loss in binding affinity, but this
was regained following optimisation, and selectivity against a
human kinase panel was improved. Although the best of these
Table 4
SAR of trisubstituted pyrimidines with functionalised phenyl ring portion
Compound NR1R2
R3
PknB IC50
Ma
/
M. tuberculosis
MIC/
l
l
Ma
N
15a
CN
0.056
63
N
N
15b
CN
CN
0.039
0.023
63
31
63
N
H
N
15c
N
H
N
15d
SO2Me 0.040
N
H
a
Assay conditions are described in Ref. 8.
number of human kinases,10 so achieving selectivity in these com-
pounds was therefore a key consideration and their kinase selectiv-
ity against a 76-member human kinase panel was examined.
Pleasingly compound 16c, containing the aminopyridine head
group motif, showed an improvement in selectivity relative to
11e, which contains the aminopyrazole head group (Fig. 2). This
is consistent with an expected gain in selectivity that may occur
through reducing the number of H-bonds that can be formed with
the kinase hinge region. Docking studies were performed using
Glide™ (Schrödinger Inc.) on both the aminopyrazole and
compounds still only showed whole cell MICs in the 10 lM region,
this level of activity is commensurate with that reported for a
number of other heterocyclic compounds which have shown activ-
ity against M. tuberculosis.13 Possible reasons for the significant
difference in enzyme and whole cell activity were investigated,
including poor cell wall permeability, the role of efflux pumps,
H
H
H
N
N
N
N
N
N
N
N
HN
HN
N
a
Cl
N
HN
c
b
N
N
R3
R1R2N
N
S
R1R2N
Cl
S
Cl
N
S
12
13
14
15a-d
Scheme 4. Reagents and conditions: (a) 3-Amino-5-cyclopropylpyrazole, Et3N, NaI, DMA, microwave, 150 °C; (b) R1R2NH, n-BuOH, microwave, 170 °C; (c) R3PhB(OH)2,
Pd(PPh3)4, copper(I) thiophene carboxylate, dioxane, microwave, 150 °C.

T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3349–3353
3353
Figure 2. Kinase selectivity heat map comparing compounds 11e (top) and 16c (bottom) against a 76-member human kinase panel, screened at 10
lM inhibitor
concentration. [Key: red: >90% inhibition; yellow: 70–90% inhibition; green: <70% inhibition.11
]
Figure 3. Example aminopyrazole 15c (left) and aminopyridine 16d (right) head group compounds docked into the PknB active site.
I.; Nemeth, G.; Hegymegi-Barakonyi, B.; Eros, D., et al Immunol. Lett. 2008, 116,
225.
6. Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64, 29.
7. Villarino, A.; Duran, R.; Wehenkel, A.; Fernandez, P.; England, P.; Brodin, P.;
Cole, S. T.; Zimny-Arndt, U.; Jungblut, P. R.; Cerveñansky, C.; Alzari, P. M., et al J.
Mol. Biol. 2005, 350, 953.
protein binding in the assay media and inhibitor specificity, and
this work is described in a separate paper.8
Acknowledgements
8. Lougheed, K. E. A.; Osborne, S. A.; Saxty, B.; Whalley, D.; Chapman, T.; Bouloc,
N.; Chugh, J.; Nott, T. J.; Patel, D.; Spivey, V.; Kettleborough, C. A.; Bryans, J. S.;
Taylor, D. L.; Smerdon, S. J.; Buxton, R. S. Tuberculosis 2011, 91, 277.
9. Liebeskind, L. S.; Srogl, J. Org. Lett. 2002, 4, 979.
10. See for example: (a) Humphries, P. S.; Lafontaine, J. A.; Agree, C. S.; Alexander,
D.; Chen, P.; Do, Q.-Q. T., et al Bioorg. Med. Chem. Lett. 2009, 19, 2099; (b)
Ioannidis, S.; Lamb, M. L.; Wang, T.; Almeida, L.; Block, M. H.; Davies, A. M., et al
J. Med. Chem. 2011, 54, 262; (c) Lin, W.-H.; Hsieh, S.-Y.; Yen, S.-C.; Chen, C.-T.;
Yeh, T.-K.; Hsu, T., et al Bioorg. Med. Chem. 2011, 19, 4173; (d) Harrington, E. A.;
Bebbington, D.; Moore, J.; Rasmussen, R. K.; Ajose-Adeogun, A. O.; Nakayama,
T., et al Nat. Med. 2004, 10, 262.
11. Kinase selectivity profiling was carried out at the National Centre for Protein
Kinase Profiling in the MRC Protein Phosphorylation Unit at the University of
Dundee.
12. Wehenkel, A.; Fernandez, P.; Bellinzoni, M.; Catherinot, V.; Barilone, N.;
Labesse, G.; Jackson, M.; Alzari, P. M. FEBS Lett. 2006, 580, 3018 (PDB ref:
2FUM).
We thank Dony Patel, Tim Nott and Vicky Spivey for production
of PknB enzyme and GarA substrate. This work was supported by
Grants from the MRCT development gap fund (award A853-0058)
and the MRC (U117585867 and U117584228).
References and notes
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