Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency, selectivity and structural novelty

Article abstract of DOI:10.1016/j.bmcl.2019.08.014

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.

Full text of DOI:10.1016/j.bmcl.2019.08.014

Journal Pre-Proof
Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: ap-
proaches to combining improvements in cell potency, selectivity and structural
novelty
Jonathan M. Large, Kristian Birchall, Nathalie S. Bouloc, Andy T. Merritt, Ela
Smiljanic-Hurley, Denise J. Tsagris, Mary C. Wheldon, Keith H. Ansell, Peter
J. Coombs, Catherine A. Kettleborough, David Whalley, Lindsay B. Stewart,
Paul W. Bowyer, David A. Baker, Simon A. Osborne
PII:
S0960-894X(19)30541-4
https://doi.org/10.1016/j.bmcl.2019.08.014
BMCL 26610
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 July 2019
7 August 2019
8 August 2019
Please cite this article as: Large, J.M., Birchall, K., Bouloc, N.S., Merritt, A.T., Smiljanic-Hurley, E., Tsagris, D.J.,
Wheldon, M.C., Ansell, K.H., Coombs, P.J., Kettleborough, C.A., Whalley, D., Stewart, L.B., Bowyer, P.W., Baker,
D.A., Osborne, S.A., Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to
combining improvements in cell potency, selectivity and structural novelty, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.08.014
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Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to
combining improvements in cell potency, selectivity and structural novelty
Jonathan M. Large,a,* Kristian Birchall,a Nathalie S. Bouloc,a Andy T. Merritt,a Ela Smiljanic-
Hurley,a Denise J. Tsagris,a Mary C. Wheldon,a Keith H. Ansell,a Peter J. Coombs,a Catherine
A. Kettleborough,a David Whalley,a Lindsay B. Stewart,b Paul W. Bowyer,b David A. Baker,b
Simon A. Osborne.a
a Centre for Therapeutics Discovery, LifeArc, Accelerator Building, Open Innovation Campus,
Stevenage, SG1 2FX, U.K.
b Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine,
Keppel Street, London, WC1E 7HT, U.K.
Graphical Abstract
Abstract
Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic
GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards
desirable in vitro property space. LLE-based approaches towards combining improvements in
cell potency, key physicochemical parameters and structural novelty are described, and a
structure-based design hypothesis relating to substituent regiochemistry has directed efforts
towards key examples with well-balanced potency, ADME and kinase selectivity profiles.
Keywords: Malaria, Plasmodium falciparum, cGMP, Protein Kinase G, Imidazopyridine,
SAR
Malaria is one of the most prevalent infectious diseases of the developing world in
humans, whose causative agent is the protozoan parasite Plasmodium, with most deaths caused
by P. falciparum. Despite being largely preventable and treatable, it was responsible for
435,000 deaths in 2017; young children and pregnant women in sub-Saharan Africa are
particularly at risk.1 In addition to continuing challenges in the contexts of policy development
and socio-economic impact,2 the observation of increasing resistance to current standard-of-
care treatments is significant. This is driving research and development efforts to uncover new
mechanisms by which the disease can be controlled and prevented.3

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Studies on the malarial kinome continue to provide well characterised and credible new
targets for antimalarial small molecule drug discovery.4,5 The cGMP-dependent kinase PfPKG
is one kinase which meets many of the criteria for such a target. Pharmacological
characterisation using early chemical inhibitors in combination with reverse genetics has
demonstrated the important role of this enzyme in numerous critical processes in the malaria
life cycle.6-11 Following previous experience with progressing chemical inhibitors of other
important malarial kinases,12-15 we have recently begun to disclose our efforts to develop a
series of PfPKG inhibitors based upon both bicyclic16 and monocyclic scaffolds.17 In the
bicyclic series, a number of advanced analogues were shown to possess promising in vitro
activity, a well-defined mechanism of action and property profiles which translated to target-
driven efficacy in vivo.16 An ongoing objective is to develop this chemical series with a view
to improving key physiochemical parameters and compound novelty whilst retaining cell
potency and lipophilic ligand efficiency (LLE).19
A recent report from us described initial efforts towards these goals by evaluating the
aminopyrimidine hinge binding motif, bicyclic core structure and basic substituent
positioning.20 Investigation of each of those structural features was found to be both necessary
and productive, and the resulting compound profiles pointed strongly to retaining these motifs
in their original forms. As a result, the profiles of analogues such as 1 (Figure 1) challenged us
to consider additional strategies for re-positioning the series in suitable ADME property space
whilst maintaining suitable levels of in vitro activity and improving compound novelty. A first
approach was to reduce the size of the 4-fluorophenyl motif to lower lipophilicity and hence
increase lipophilic ligand efficiency (LLE)19 (Figure 1 – A). Previous SAR20 suggested potency
could be regained by enlarging the pyrimidine substituent, if needed (Figure 1 – B). In a second
set of analogues, it was anticipated that re-design of the benzylic dimethylaminomethyl side
chain in the prototypical inhibitor compound 2 would enable lowering of logD (for example
by increasing chain length and basicity) and could also address one likely point of metabolic
liability (for example by replacing the benzylic carbon atom with a heteroatom) (Figure 1 - C).
Here we discuss the results of these investigations and show their significant beneficial impact
against the above criteria.
Figure 1. In vitro profiles of imidazopyridine 1 and 2, and design modifications to be applied to 2: A – truncate
the aryl group; B – enlarge the pyrimidine substituent if required; C – re-design the basic substituent. ADME data:
mLogD = measured logD; MLM = % remaining after 30 min incubation with mouse liver microsomes.
We first examined the possibility of improving the lipophilic efficiency by focusing on
the large 4-fluorophenyl motif, and initially retained the original basic substituent at the 7-
position of the bicyclic core in doing so. The main design emphasis was to attempt to balance
the size of the pyrimidine substituent with a smaller lipophilically efficient replacement for the
4-fluorophenyl group. Among a small set of initial replacements, prepared by the general route
shown in scheme 1, the cyclopropyl analogue 9 was of lower potency in a biochemical assay21
as compared to 2, but significantly also showed a lower mLogD value of 1.7 of 1.7 (Table 1).22
Given that potency was lower than desirable, further analogues incorporating the cyclopropyl

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group were designed to combine a lower mlogD with improvements in potency and LLE.
Hence a set of compounds with larger groups appended to the aminopyrimidine nitrogen was
prepared using variations of the same chemical approach. Small alkyl groups such as that in 10
did not provide any further boost in activity or LLE but, in line with previous SAR,
arylaminopyrimidines such as 11 and 12 were more biochemically active and possessed the
anticipated trend towards lower mLogD. The most balanced profilewas achieved in 13,23 which
showed similar levels of both biochemical potency and anti-malarial activity in a blood stage
hypoxanthine incorporation (HXI) cell assay21 compared to 2, coupled with improvements in
mLogD and LLE.
Scheme 1. Reagents and conditions: (i) LiHMDS, R1CO2Et, THF, -78 oC - rt, 3 h, 27-76%; (ii) Bu4NBr3 or NBS,
o
CH2Cl2, rt, 2 h; (iii) 2-aminopyridine-4-methanol, EtOH, 4Å sieves, 100 C, 18 h, 12-44% for two steps; (iv)
MsCl, Et3N, THF, 0 oC, 1 h or SOCl2, CH2Cl2, 50 oC, 1 h; (v) Me2NH, THF, 0 oC – rt, 33-65% for two steps; (vi)
o
H2O2, Na2WO4.2H2O, AcOH, MeOH, 0 C – rt, 3 h; (vii) for 7 – 9: NH4OAc, melt, 130 oC, 3 h, 5-27% for two
o
steps; for 10: iPrNH2 (neat), 60 C 3 h, 23% for two steps; for 11: 2-aminopyridine (excess), NMP, microwave,
o
o
150 C, 3 h, 8% for two steps; for 12: 4-(4-methylpiperazino)aniline, neat, microwave, 170 C, 15 min, 5% for
two steps; for 13: 4-(4-aminophenyl)piperazine-1-carboxylic acid tert-butyl ester, TFA, sBuOH, 110 oC, 6 h, then
TFA, CH2Cl2, rt, 2 h, 10% for three steps.

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Table 1. Replacing the 4-fluorophenyl group with a cyclopropyl motif
PfPKG Pf HXI
mLog
D
Compound
R1
LLE
6.3
a
pIC50
pEC50
2
-
8.70
6.44
2.4
1.7
9
H
7.45
7.36
nt
nt
5.8
4.4
10
3.0
2.5
11
12
8.06
8.14
5.76
6.95
5.8
6.0
2.1
1.5
13
8.35
6.70
6.8
a nt = not tested.
Turning next to the basic substituent on the bicyclic core, a small number of molecules
were initially designed to identify the optimum position at which to locate this motif. We
decided to employ the benzylic dimethylaminomethyl group present in 2 for this analysis.
Docking of 2 and its 5-, 6- and 8- regioisomers into an apo-structure of PfPKG (PDB:5DYK24)
suggested that the best site for that substituent was at the 7-position (Figure 2). The location of
the positively charged basic center between two acidic protein residues (E625 and D682) was
judged to be optimal for that particular group. Whilst relocating to the 6- or 8- positions
appeared to be spatially tolerable, sub-optimal interaction with the acids and a subsequent loss
in affinity was predicted. Appending several possible groups at the 5-position appeared to result
in a significant steric clash with the pyrimidine hinge binding motif (data not shown); this was
predicted to cause a significant loss of activity and hence was not pursued.
Figure 2. Docking of 2 (left), the 8-regioisomer 15 (centre) and the 6-regioisomer 16 (right) into an apo-PfPKG
crystal structure (PDB:5DYK24), with protein surface coloured by electrostatic potential. H-bonds and charge
interactions are shown as dashed lines.

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This hypothesis was tested by synthesizing the 8- and 6-regioisomers 15 and 16
respectively. Using variations of previously described chemical approaches,18,20 compounds 15
and 16 could be prepared from the bromoketone building block 1425 (Scheme 2), in good yields
over 5 synthetic steps.
Scheme 2. Reagents and conditions: (i) 2-aminopyridine-3-methanol (for 15) or 2-aminopyridine-5-methanol (for
16), MeCN, NaHCO3, 90 oC, 18 h: 32-75%; (ii) MsCl, Et3N, THF, 0 oC, 4 h; (iii) Me2NH, THF, 0 oC - rt, 3 h, 60-
89% for two steps; (iv) H2O2, Na2WO4.2H2O, AcOH, MeOH, rt, 3 h; (v) NH4OAc, melt, 120 oC, 3 h, 19-27% for
two steps.
These two compounds showed reductions in their biochemical activity, as compared to
2 (Table 2), which were in line with predictions from the docking studies. Lipophilic ligand
efficiency for the 8-substituted compound 15 was also higher than for 6-analogue 16, in part
due to an interesting divergence in mLogD (values of 1.6 for 15, 2.3 for 16, as compared to 2.4
for 2). However, the key factor of lower synthetic accessibility for 8-position analogues
emerged, which directed our efforts away from preparing further compounds of this kind. In
contrast, the position of the two key acidic residues at the binding pocket mouth implied that
re-design of the basic substituent into longer chain variants and appropriate conformationally
constrained versions might be productive. This design hypothesis suggested that substituents
of these new types at either the 6- or 7-positions should be evaluated.
Table 2. Position of basic substituent attachment
Substituent
position
PfPKG
pIC50
Compound
LLE
6.3
2
7
8
6
8.70
7.53
7.29
15
16
5.9
5.0

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We tested this proposal by making compounds bearing such modifications to the
dimethylaminomethyl side chain in 2, and chose to include adjustments in both expected pKa
and conformation by varying the linking atom, chain length and ring size in new analogues.
Preparation of key intermediates 17 – 19 in three steps, followed by palladium-catalysed
aminations or microwave-mediated direct displacements provided the N-linked and O-linked
examples 20 – 27 respectively (Scheme 3).26 The 7-C-linked analogue 29 was also prepared in
four synthetic steps from intermediate 28, which was itself constructed by condensing
bromoketone 14 with the appropriate building block 2-(2-aminopyridin-4-yl)ethan-1-ol.
Scheme 3. Reagents and conditions: (i) 2-amino-4-bromopyridine or 2-amino-5-bromopyridine, EtOH, 4Å sieves,
80 oC, 18 h, 20-36%; (ii) H2O2, Na2WO4.2H2O, AcOH, MeOH, rt – 50 oC, 18 h; (iii) For R1 = H: NH4OAc, melt,
o
o
130 C, 18 h, 30-52% for two steps; for R1 = Me: Me2NH, THF, 70 C, 18 h, 56% for two steps; (iv) for R2 =
amine: Pd(OAc)2, JohnPhos, R2NH, NaOtBu, dioxane, 100 oC, 18 h, 3-29%; (v) for R2 = alcohol: KOtBu, ROH,
NMP, microwave, 170 oC, 10 min, 6%; (vii) MsCl, Et3N, THF, 0 oC, 1 h; (vi) Me2NH, THF, 60 oC, 10 h, 49% for
two steps.
These analogues (along with the earlier example 16) demonstrated that extending via a
conformationally constrained basic group (as in 20) or via an open chain version (as in 21) at
either the 7- or 6-position of the core could each provide good biochemical potency (Table 3).
Neither of these compounds appeared to possess a particular advantage in any aspect of their
in vitro profiles as compared to 2. Interestingly, the related pair of piperazine regioisomers 22
and 23 showed a subtle contrast in mLogD, where the 6-isomer 23 was found to possess the
lower value. The cell activity of 23 was also slightly lower as compared to 22. Synthetic access
to 6-substituted compounds was also found to be generally less efficient; considering this and
other contributing factors,27 we decided to focus additional efforts on 7-linked analogues only.

Table 3. Basic side chain variationsJOURNAL PRE-PROOF
PfPKG
pIC50
Pf HXI
pEC50
Compound
R1
LLE mLogD
a
2
7-CH2NMe2
8.70
7.29
6.44
6.3
5.0
2.4
2.3
16
20
6-CH2NMe2
nt
7-
8.07
6.07
6.0
2.1
21
22
6-NH(CH2)3NMe2
7-
7.90
8.48
nt
5.7
5.4
2.2
3.1
6.58
6-
23
8.58
6.06
5.8
2.8
a nt = not tested.
Using the same synthetic chemistry as shown in Scheme 3, a small additional set of 7-
substituted analogues was prepared and evaluated (Table 4). As compared to 20, increasing the
ring size and hence altering the conformational constraint in 24 gave modest improvements in
biochemical potency and cell activity, though only a slight change to LLE. Microsomal stability
was improved significantly, perhaps due to constraining the conformation in the basic side
chain. For the open chain examples 25 and 29, in vitro ADME profiles very similar to 2 could
be obtained, though both showed lower biochemical activity (and hence no further benefit in
LLE) and microsomal stability had not improved. Both LLE and microsomal stability could be
improved by returning to a nitrogen-linked design in the open chain analogue 26, for which the
essentially unchanged mLogD (as compared to 25 and 29) was accompanied by better
biochemical potency and LLE. Finally, positioning an additional carbon atom within the
aminopyrimidine group gave 27; this notable compound showed an excellent balance of good
biochemical potency, in vitro activity against the parasite and improved LLE and mLogD
values. The effect of the secondary aminopyrimidine (in 27) on microsomal stability, relative
to the primary aminopyrimidine (in 26), was also significant.

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Table 4. Basic substituents at the 7-position
PfPKG Pf HXI
MLM
Compound
R1
R2
H
LLE
6.3
mLogD
2.4
a
pIC50
pEC50
% remb
2
7-CH2NMe2
8.70
6.44
52
88
24
H
8.44
6.46
6.2
2.2
25
26
27
29
7-O(CH2)2NMe2
7-NH(CH2)2NMe2
7-NH(CH2)2NMe2
7-(CH2)2NMe2
H
H
8.13
8.59
8.56
7.69
6.09
nt
5.9
6.5
6.4
5.8
2.2
2.1
2.2
1.9
54
80
93
65
Me
H
6.28
6.00
a nt = not tested; b % remaining after 30 min incubation with mouse liver microsomes.
The two most promising compounds identified - 13 and 27 - were profiled and
compared in vitro (Table 5). In addition to previously described improvements in mLogD and
LLE, high kinetic solubility (measured using PBS at pH7.4 as buffer) was maintained in each
case and both compounds were shown to be non-cytotoxic. Despite a significantly lower
mLogD value, the mouse microsomal stability of 13 surprisingly remained at the same level as
for 2, for which we have no clear explanation.28 In particular, 27 matched excellent biochemical
and cell potency with significantly higher stability in mouse microsomes to give a highly
promising and well-balanced overall profile. Selectivity was assessed by screening 13 and 27
against a human kinase panel29 at a single 1 µM concentration (Figure 3). As expected, the
smaller cyclopropyl motif in 13 resulted in a decreased level of selectivity, whilst 27 showed
an excellent selectivity profile against the kinases screened. We also tested compounds 2 and
27 against the two human orthologues of PKG; no activity was observed up to a top assay
concentration of 1 µM,30 indicating a high level of selectivity for the malarial kinase.
a
Table 5. Full in vitro profiles for compounds 2, 13 and 27; % remaining after 30 min incubation with mouse
liver microsomes; b kinetic solubility; c in vitro cytotoxicity assay measured in HepG2 human liver-derived cells
– concentration at which half of cells remained viable at 48 h.16
PfPKG Pf HXI
MLM
Kin sol HepG2
Compound
LLE
mLogD
c
pIC50
pEC50
% rema
(µM)b
pEC50
2
8.70
8.35
8.56
6.44
6.70
6.28
6.3
6.8
6.4
2.4
1.5
2.2
52
53
93
200
189
207
< 4.7
< 5
13
27
< 4.7

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Figure 3. Kinase selectivity data for representative imidazopyridines 2, 13 and 27 on screening against a human
kinase panel at 1 µM concentration; green < 50% inhibition; yellow 50-90% inhibition; red > 90% inhibition.29
We have reported here the results of our continuing effort to progress a series of
imidazopyridines as inhibitors of PfPKG, focusing on alteration of the 4-fluorophenyl group
and re-design of the basic substituent as key strategic aims. By concentrating on cell potency,
lipophilic ligand efficiency and structural novelty in tandem, compounds such as 27 in
particular were developed to populate a highly desirable and novel area of chemical space as
potent, lower molecular weight, lipophilically efficient analogues with improved in vitro
ADME and selectivity profiles. Studies towards the identification of additional analogues
suitable for in vivo studies and further mechanistic considerations are ongoing and will be
reported in due course.
Acknowledgments
This work was funded by MRC DPFS grant no G1000779. The authors thank David
Tickle, Sadhia Khan, Katie Barnes and Hasina Mahmood (LifeArc) for compound purification
and in vitro ADME data, Win Gutteridge and Simon Croft for many helpful discussions, and
Jeremy Burrows and Sir Simon Campbell (Medicines for Malaria Venture) for their support of
this work.
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21. Full details on biochemical assay and anti-malarial hypoxanthine incorporation (cell
based) assay procedures can be found in reference 16.
22. Installing either a methyl group (7; PfPKG pIC50 7.24) or trifluoromethyl group (8; PfPKG
pIC50 6.83) gave intermediate levels of biochemical potency.
23. For an improved synthetic approach to compound 13, see supplementary information.
24. Wernimont, A.K.; Tempel, W.; He, H.; Seitova, A.; Hills, T.; Neculai, A.M.; Baker, D.A.;
Flueck, C.; Kettleborough, C.A.; Arrowsmith, C.H.; Edwards, A.M.; Bountra, C.; Hui, R.;
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25. Scribner, A.; Dennis, R.; Hong, J.; Lee, S.; McIntyre, D.; Perrey, D.; Feng, D.; Fisher, M.;
Wyvratt, M.; Leavitt, P.; Liberator, P.; Gurnett, A.; Brown, C.; Mathew, J.; Thompson, D.;
Schmatz, D.; Biftu, T. Eur. J. Med. Chem. 2007, 42, 1334.
26. Low yields for the final palladium-catalysed reactions could be improved by reversing the
order of synthesis, such that this step was carried out before oxidation and displacement
of the 2-thiomethylpyrimidine substituent. Full details are provided in the supplementary
information.
27. It was found that 23 showed significant activity in an in vitro cytotoxicity assay16 whereas
22 did not. The same relationship between cell cytotoxicity and regiochemistry of the basic
substituent on the bicyclic core was also observed for earlier compounds 16 and 2.
28. For an excellent recent review on correlating ADME properties, see Kramer, C.; Ting, A.;
Zheng, H.; Hert, J.; Schindler, T.; Stahl, M.; Robb, G.; Crawford, J. J.; Blaney, J.;
Montague, S.; Leach, A. G.; Dossetter, A. G.; Griffen, E. J. J. Med. Chem. 2018, 61, 3277.
29. Kinase selectivity profiling was carried out at the MRC Protein Phosphorylation Unit at
the University of Dundee, U.K. at a single concentration of 1 µM for all three compounds.
Kinases tested are listed below (reading from left to right in Figure 3) – the two kinases
against which all three compounds showed some activity are in bold type:
MKK1, ERK1, ERK2, JNK1, JNK2, p38a, MAPK, RSK1, RSK2, PDK1, PKBa, PKBb,
SGK1, S6K1, PKA, ROCK2, PRK2, PKCa, PKCz, PKD1, MSK1, MNK1, MNK2,
PRAK, CAMKKb, CAMK1, SmMLCK, PHK, CHK2, GSK3b, CDK2-Cyclin A, PLK1,
Aurora A, Aurora B, AMPK, MARK3, BRSK2, MELK, CK1, CK2, DYRK1A, NEK2a,
NEK6, IKKb, PIM1, SRPK1, MST2, EF2K, HIPK2, PAK4, Src, Lck, CSK, FGF-R1, IRR,
MST4, SYK, YES1, IGF-1R, VEG-FR, BTK, EPH-B3, TBK1, IKKe, GCK, NUAK1,
MLK1, MINK1, MLK3, LKB1, HER4, TTK, IR, RIPK2, TAK1, MEKK1, TrkA.
30. For additional details see supplementary information.