Discovery of selective, orally bioavailable pyrazolopyridine inhibitors of protein kinase cθ (pkcθ) that ameliorate symptoms of experimental autoimmune encephalomyelitis

Article abstract of DOI:10.1021/acsmedchemlett.9b00134

PKCθ plays an important role in T cell biology and is a validated target for a number of disease states. A series of potent and selective PKCθ inhibitors were designed and synthesized starting from a HTS hit compound. Cell activity, while initially a challenge to achieve, was built into the series by transforming the nitrile unit of the scaffold into a primary amine, the latter predicted to form a new hydrogen bond to Asp508 near the entrance of the ATP binding site of PKCθ. Significant improvements in physiochemical parameters were observed on introduction of an oxetane group proximal to a primary amine leading to compound 22, which demonstrated a reduction of symptoms in a mouse model of multiple sclerosis.

Full text of DOI:10.1021/acsmedchemlett.9b00134

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Letter
Discovery of Selective, Orally Bioavailable Pyrazolopyridine
Inhibitors of Protein Kinase C# (PKC#) that Ameliorate
Symptoms of Experimental Autoimmune Encephalomyelitis
Philip N Collier, Heather C. Twin, Ronald M. A. Knegtel, Dean Boyall, Guy Brenchley, Christopher
J. Davis, Shazia Keily, Chau Mak, Andrew Miller, Françoise Pierard, Luca Settimo, Clare M.
Bolton, Peter Chiu, Adam Curnock, Elisabeth Doyle, Adam J Tanner, and Juan-Miguel Jimenez
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00134 • Publication Date (Web): 27 Jun 2019
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Discovery of Selective, Orally Bioavailable Pyrazolopyridine
Inhibitors of Protein Kinase Cθ (PKCθ) that Ameliorate Symptoms
of Experimental Autoimmune Encephalomyelitis
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Philip N. Collier,*† Heather C. Twin,* Ronald M. A. Knegtel, Dean Boyall, Guy Brenchley,
Christopher J. Davis, Shazia Keily, Chau Mak, Andrew Miller, Françoise Pierard, Luca Settimo, Clare
M. Bolton, Peter Chiu, Adam Curnock, Elisabeth Doyle,† Adam J. Tanner, Juan-Miguel Jimenez
Vertex Pharmaceuticals (Europe) Ltd., 86-88 Jubilee Avenue, Milton Park, Abingdon OX14 4RW, United Kingdom
†Vertex Pharmaceuticals Inc., 50 Northern Avenue, Boston, Massachusetts 02210, United States
KEYWORDS. PKCθ, multiple sclerosis, kinases, oxetane.
ABSTRACT: PKCθ plays an important role in T cell biology and is a validated target for a number of disease states. A series of
potent and selective PKCθ inhibitors were designed and
synthesized starting from a HTS hit compound. Cell activity,
while initially a challenge to achieve, was built into the series by
transforming the nitrile unit of the scaffold into a primary amine,
the latter predicted to form a new hydrogen bond to Asp508 near
the entrance of the ATP binding site of PKCθ. Significant
improvements in physiochemical parameters were observed on
PKCθ Ki = 3 nM
IL-2 IC50 = 2.6 μM
PKCθ Ki = 6 nM
IL-2 IC50 = 0.19 μM
introduction of an oxetane group proximal to a primary amine
leading to compound 22, which demonstrated a reduction of
symptoms in a mouse model of multiple sclerosis.
Efficacy in murine EAE
Protein kinase C (PKC) are a large family of serine/threonine
kinases that are involved in the regulation of a number of
essential cellular processes. The family consists of 11 PKC
isoforms that are separated into 3 different classes: (i)
diacylglycerol (DAG) and calcium dependent classical
isoforms (PKCs α, βI, βII, and γ); (ii) DAG-dependent novel
isoforms (PKCs δ, ε, η, and θ); (iii) DAG and calcium
independent atypical isoforms (PKCs λ, ι, and ζ).1 PKCθ, one
of the novel PKC isoforms, mediates an immune response via
T-cell activation, differentiation and migration and is the only
member of the PKC family known to translocate to the
immunological synapse.2
to be important for the development of T cell mediated
inflammatory diseases, because PKCθ-deficient mice display
resistance in models of asthma5 and IBD,6 collagen-induced
arthritis
(CIA),7
and
experimental
autoimmune
encephalomyelitis (EAE), a commonly used model of multiple
sclerosis (MS).8,9 PKCθ-deficient mice however, are able to
mount normal T-cell responses to certain pathogens, and anti-
viral responses seem to remain intact.10-12 In addition to the
unique role that PKCθ plays in T-cell activation, inhibition of
PKCθ enhances Treg function.13 PKCθ inhibition may be a
useful
approach
towards
treating
T-cell-mediated
inflammatory diseases such as rheumatoid arthritis,
inflammatory bowel disease and psoriasis, by inhibiting T-cell
activation and enhancing Treg function, without leading to
broad immunosuppression.
PKCθ is mainly expressed in T lymphocytes and the
phenotype of the knockout mouse implies a central role in T-
cell activation by integrating signals from both the T cell
receptor (TCR) and the co-stimulatory CD28.3,4 PKCθ appears
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Despite the PKC family being the focus of research over the
Cpda
ROCK,
JAK2
Cl/Fu
Rat iv PKc
Cl, T1/2, Vss
PKCθ
PKC α,
(R/H)b
past two decades,14,15 a selective PKCθ inhibitor has yet to
reach the clinic.16-18 The catalytic domain is highly conserved
among the different isoforms thus making the design of
1
2
3
4
5
6
7
8
δ
1
2
3
3
110, 12
465, 240
ND, 584
6, 13
156, 24
24, 33
42, 0.3,
0.99
selective inhibitors challenging.19 Sotrastaurin (AEB071),20
a
pan-PKC inhibitor developed by Novartis, inhibits T-cell
activation via PKC by the binding of peptide-MHC complexes
and CD28 co-stimulation.21 Sotrastaurin displays poor kinome
selectivity, inhibiting more than 200 other kinases, including
those important for early T-cell activation such as Lck. A
transient increase in heart rate has been observed in clinical
trials with Sotrastaurin, leading to questions around the
feasibility of developing a safer approach through selective
inhibition of one PKC isoform instead of multiple PKC
isoforms.22,23 Selective PKCθ inhibition is postulated to
provide a better balance of efficacy and safety.
14
28
210, 86
560, 270
22, 2.5,
1.22
58, 172
12, 4.6,
0.94
aAll enzyme data are Ki and expressed in nM. Unbound Hep
Cl expressed in μL/min/106cells. CL expressed in mL/min/Kg,
b
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T1/2 in h and Vss in L/Kg.
Compound 1 was docked in a published crystal structure of
PKCθ as described previously.27 In the resulting model, the
cyano moiety sits in a small hydrophobic pocket under the
glycine-rich loop (GRL) (Figure 1). Opportunities for
obtaining selectivity over PKC isoforms were identified by
comparing the model of compound 1 bound to PKCθ with
homology models of other PKC isoforms built using PKCθ as
a structural template. Within the C-terminus there is a
significant sequence difference between the PKC isoforms,
involving the replacement of the relatively small Cys661 in
PKCθ with much larger residues such as Tyr630 in PKCδ
(Figure 1). As a consequence of this residue difference, a
larger hydrophobic pocket, delineated by Phe664 (C-terminus)
and Leu386 (GRL), is accessible in PKCθ as compared to
other PKCs. This area of the ATP binding site could be further
explored to gain both potency and selectivity.
In 2015, AbbVie published their efforts towards the discovery
of selective PKC theta inhibitors.24,25 An advanced lead
compound displayed efficacy in a mouse model of arthritis but
was poorly tolerated at doses marginally higher than the
efficacious dose. Further efforts focused on improving
tolerability, led to compounds that, despite good exposure,
yielded only moderate efficacy in a chronic in vivo mouse
model of arthritis.
As part of our own program to identify potent and selective
PKCθ inhibitors,26-28 biochemical screening of the Vertex
compound collection led to the identification of 1 as a suitable
starting point for medicinal chemistry optimization efforts
(Table 1). It displayed high affinity for inhibition of PKCθ and
possessed drug-like properties (MW 262, clogP 2, PSA 65
Å2). Compound 1 showed some isoform selectivity against
PKCα but minimal selectivity against PKCδ, an enzyme
deemed necessary to avoid because of the risk of B-cell
autoimmunity associated with its inhibition. It also showed
strong activity against a range of other kinases (e.g. Ki: PKA,
110 nM; GSK-3β, 210 nM; ROCK, 6 nM; JAK-3, 3 nM; FLT-
3, 100 nM). Poor rat hepatocyte stability resulted in high
clearance when 1 was dosed intravenously in rat.
Initial optimization work led to improvements in general
kinase selectivity as a result of transitioning from a 1H-
pyrrolo[2,3-b]pyridine to a 1H-pyrazolo[3,4-b]pyridine core
(Table 1). The selectivity improvements observed with 2 and
3 are likely due to the presence of Thr442, located directly
underneath the pyrazolopyridine 2-position nitrogen atom,
being replaced by a more lipophilic valine in other kinases like
ROCK, PKA and JAK2. These modifications also led to
improved rat metabolic stability and lower in-vivo clearance.
The improved rat hepatocyte stability of 2 compared to 1,
correlates with a lower lipophilicity (logD7.4 of 1, 3.4; of 2,
2.9).
Figure 1. Predicted binding mode of 1 (cyan) in PKCθ
(grey/green). The cyano group points into a small hydrophobic
pocket under the glycine-rich loop (GRL). The replacement of the
smaller Cys661 in PKCθ with the larger Tyr630 in PKCδ (brown)
in the C-terminus results in the presence of a larger hydrophobic
pocket in PKCθ located between Phe664 (C-terminus) and
Leu386 (GRL)
TABLE 1. Early Optimization of HTS Hit Compound 1.
Based on the above modeling, a range of α,α-disubstituted
nitriles were designed and synthesized, all possessing
nanomolar biochemical affinity (data not reported). However,
none of these compounds or the ones reported earlier in the
manuscript had sub-micromolar cell potency for the inhibition
of IL-2 release from PBMCs following stimulation with anti-
CD3/CD28. It was confirmed that poor cell permeability was
not the reason behind the observed weak cell potency (Caco-2
data for compound 2: A-B = 20.1 x 10-6 cm/s, E.R. = 0.95;
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compound 3: A-B: 36 x 10-6 cm/s, E.R. = 0.7). A breakthrough
1
2
in this area came when the nitriles were converted into the
Table 2. SAR exploration of scaffold benzylic position
corresponding primary amines (Table 2, 5 - 11).
3
4
5
6
7
8
9
R1 R2
Molecular modeling studies suggested a different binding
mode of amine 6 in PKCθ compared to nitrile 4 (Table 2).
When docked into PKCθ, the primary amine of 6 projects
towards solvent, unlike the nitrile group of 4 which projects
under the glycine-rich loop. This change in positioning of the
polar functionality is driven by a predicted hydrogen bond
involving the amine of 6 and the backbone carbonyl of
Asp508 as well as favorable electrostatic interactions with the
carboxylate groups of aspartates 465 and 508 as shown in
Figure 2.
R1 R2
CN
NH2
N
N
N
H
N
H
N
N
5 - 11
10
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Cpd
R1, R2
PKCθ
IL-2
PKCδ / PKCα /
Kia
b
IC50
PKCθ
PKCθ
5
6
Me, Me
Me, Et
12
3
16
13
36
18
18
8
50
37
0.33
0.17
0.02
0.30
0.35
0.58
0.55
7
Me, CH2CCH
-(CH2)3-
0.2
17
22
30
9
149
41
8
9
-(CH2)4-
28
10
11
-(CH2)5-
18
-(CH2)2O(CH2)2-
111
93
aEnzyme Ki are expressed in nM. Cell IC50 are expressed in
b
μM.
Figure 2. Predicted binding mode of 6 in the active site of PKCθ.
Compound 6 is coloured cyan and PKCθ is depicted in
grey/green. Key amino acid side chains are labeled and depicted
as sticks.
Cell potency could be modulated by varying the substituents at
the benzylic position of the scaffold (Table 2). Modeling
suggests that the highly potent alkyne 7 extends further into
the back of the active site and fills the same pocket occupied
by the cyano group of 4, and the ethyl group of 6 (Figure 3A).
In our model, the alkyne moiety fits tightly into this pocket
and is in van der Waals contact with the gatekeeper reside
Met458 while engaging in aromatic stacking interactions with
Phe391 of the GRL.
Figure 3. (A) Predicted binding pose of 7 in PKCθ; (B) Predicted
binding mode of 11 in PKCθ. Inhibitors are coloured cyan and
PKCθ grey/green, key amino acid side chains are labeled and
depicted as sticks.
Binding of the increasingly large alkyl rings of compounds 8-
10 requires conformational adjustments of both the inhibitor
and the kinase GRL/C-terminus. Only in PKCθ can both of
these adjustments readily be accommodated because there is
more space available between the C-terminus and the GRL.
This is because the residue equivalent to Cys661 in the C-
Selectivity over PKCδ could be improved by targeting the C-
terminus region via meta-substitution on the central benzene
ring (Table 3). As shown in Figure 1, the meta position
projects substituents towards a hydrophobic pocket located
between Phe664 (C-terminus) and Leu386 (GRL). Alkyl
substituted compounds 12-14 improved selectivity over PKCδ
terminus of PKCθ is a larger tyrosine or leucine in PKCδ and
PKCα, respectively. With the introduction of an ether oxygen
by occupying a pocket that is slightly larger in PKCθ
compared to PKCδ due to the Cys661/Tyr630 sequence
difference in the C-terminus.
in the THP ring of compound 11, modeling suggests that the
inhibitor becomes fixed in space by two hydrogen bonds
involving the salt-bridge Lys409 and the backbone carbonyl of
Asp508 (Figure 3B). With the inhibitor fixed by hydrogen
bonding, the binding of large rings now solely depends on the
ability of the GRL and C-terminus to accommodate them. The
improved selectivity profile of 11 is therefore likely due to
PKCθ having more space available near its GRL/C-terminus
interface compared to other PKC sub-types with larger
residues in their C-termini.
Table 3. SAR exploration of meta position of benzene ring
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Cpd
R
PKCθ
IL-2
Cpd
R
PKCθ
IL-2
PKCδ /
PKCθ
PKCδ /
PKCθ
Kia
Kia
b
b
IC50
IC50
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11
12
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6
H
Me
Et
3
5
14
24
39
39
0.17
0.26
0.25
0.2
6
H
Me
Cl
2
24
>16
22
0.17
0.10
0.02
0.01
12
13
14
15
16
17
<1
0.3
0.4
14
32
Pr
CF3
25
aEnzyme Ki are expressed in nM. Cell IC50 are expressed in
aEnzyme Ki are expressed in nM. Cell IC50 are expressed in
b
b
μM.
μM.
Substitution at the C3-position of the pyrazolopyridine core
led to increased PKCθ affinity. For example, substitution with
a 3-trifluoromethyl group improved cell potency ten-fold,
while maintaining a reasonable PK profile (17, rat IV PK: CL
= 45 ml/min/kg; T1/2 = 1.8 h; Vss = 2.7). As the central phenyl
ring is already rotated out of the plane of the azaindazole ring
system by ~40º due to steric hindrance with the pyrazole ring,
substitution on the 3-position only has a small effect on the
overall conformation of the inhibitor. The observed gain in
potency is presumably due to the substituent on the 3-position
occupying a small hydrophobic pocket positioned in between
the ligand’s central phenyl ring and the Met458 gatekeeper
residue of PKCθ (Figure 4).
The knowledge generated from the above SAR studies led to
the preparation of advanced lead compounds 18-22 (Table 5).
Cyclobutanes 18 and 19 potently inhibited PKCθ and secretion
of IL-2 from stimulated PBMCs, and were isoform selective
against PKCα and δ. However, they strongly inhibited the
hERG ion channel, raising safety concerns. THP compound 20
was also prepared and showed reduced activity at the hERG
ion channel. THP analogs such as 20 were deprioritized
however, due to their cross activity against other kinases (e.g.
PKA and FLT-3 Ki values, 30 and 46 nM respectively).
In order to further build on the improved profile observed with
THP analog 20, oxetane-containing compounds 21 and 22,
were prepared. It has been established that the introduction of
the oxetane unit onto molecular scaffolds can have a profound
impact on physiochemical properties such as lipophilicity,
metabolic stability and aqueous solubility.29 Although 21 and
22 displayed reduced biochemical and cellular activities, they
led to improved isoform selectivity profiles compared to 20.
21 and 22 showed improved CYP450 inhibition profiles (21:
22, >30, >30 μM vs. 18: 16, 4, 8 μM against CYP3A4, 2C9,
2D6 respectively) and generally increased metabolic stability
compared to the corresponding cyclobutanes 18 and 19,
correlating with lower logD (experimental values for 18, 21,
22: 2.9, 2.2 and 1.7 respectively). Lower hERG affinities were
observed for oxetane amines 21 and 22, when compared to
cyclobutylamines 18 and 19, perhaps reflective of the lower
basicity of the former (pKa: 7.9 and 7.6 for 21 and 22
respectively vs. 8.8 and 8.8 for 18 and 19 respectively).
Cyclobutane 19 and tetrahydropyran 20 showed cytotoxicity at
a significantly lower concentration than oxetane 22 in a HFL-1
counter-screening assay measuring inhibition of cell growth.
Figure 4. Predicted binding pose of 17 in PKCθ. The inhibitor is
colored cyan and PKCθ grey/green, key amino acid side chains
are labeled and depicted as sticks.
Oxetane 22 was chosen for further characterization in vitro
and in vivo on the basis of it possessing the most balanced
overall profile. 22 showed good selectivity when profiled
against a wide panel of kinases (inhibiting 2/49 kinases >50%
at 0.4 μM, see Supporting Information) and a non-kinase panel
of receptors and ion-channels (>50% inhibition at 10 μM for
2/68). 22 was permeable and showed no significant efflux
Table 4. SAR exploration of pyrazolopyridine C3-position
(Caco-2 A-B
= 19, ER 2.4). On intravenous (i.v.)
administration, compound 22 displayed high clearance (62
mL/min/Kg) and short half-life (1.2 h) in rat, but much lower
clearance and longer half-lives in higher species (clearance of
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12 and 9 mL/min/Kg in dog and cynomolgus monkeys
respectively; corresponding T1/2 = 6.2 and 5.1 h respectively).
22 showed moderate to good bioavailability when dosed orally
in mouse (67%) and rat (46%) and displayed good
Compound 22 was evaluated in murine experimental
autoimmune encephalomyelitis (EAE), a well-established
model of MS. Two previous studies have genetically validated
PKCθ in EAE.8,9 PKCθ knock out mice treated with MOG
antigen were resistant to EAE, the resistance reported to be
due to decreased cytokine production by T cells and failure of
these cells to penetrate into the CNS. Our group has
previously reported on the pharmacological validation of
PKCθ in EAE.28
1
2
3
4
5
6
7
8
thermodynamic solubility (515 μM). This overall PK profile
suggested that exposure of 22 would be sufficient to modulate
PKCθ in an in-vivo model. Indeed, we demonstrated that 22
significantly inhibits IL-2 as part of an assessment of IL-2
production in vivo (see Supporting Information). Furthermore,
we found that unbound brain exposure 3h post po dose was
comparable (0.19 uM @ 50 mg/kg) or exceeded (0.4 uM @
100 mg/kg) IL-2 IC50 of 0.2 uM, indicating ample target
coverage.
In this experiment, C57BL6 mice were immunized with MOG
antigen and allowed to develop symptoms of EAE. Animals
were dosed with vehicle, or 22, on reaching a clinical score of
1-1.5. We found that therapeutic dosing of 22 at 50 mg/kg was
effective in reducing symptoms in animals with ongoing
disease (Figure 5). A recent publication described the
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We also wanted to assess if compound 22 showed activity
against the Th17 subset of CD+ T-cells. There is strong
evidence that Th17 cells mediate the autoimmune
inflammation and tissue damage in MS by the release of pro-
inflammatory cytokines such as IL-17, IL-21 and IL-22. These
cells have been found to be present in both the CNS and
immune periphery in the EAE mouse model and have been
detected in the brain tissues of patients with MS.30 22
inhibited the release of IL-17 from CD3/CD28 stimulated Th-
amelioration of clinical symptoms of EAE with PKCβ
inhibitor LY-317615 via stabilization of blood-brain barrier
disruption.31 We confirmed that the efficacy we observed with
22 in EAE was not due to inhibition of the PKCβ isozyme, (Ki
= 1.7 μM for 22).
17 cells with an IC50 of 1 μM.
Table 5. Profiles of advanced lead compounds 18-22
R
PKCθ,
IL-2
HFL-1
IC50
RLM,
HLMc
hERGd
0.7
δ, α Kia IC50
b
b
3, 135,
60
0.06
0.65
56, 81
88, 81
65, 96
Figure 5. Therapeutic dosing of 22 in MOG EAE mouse model.
Please see Supporting Information for statistical analysis of these
EAE data.
2, 60,
57
0.09
0.13
1.9
3
In summary, we have discovered a series of selective
inhibitors of PKCθ starting from a promiscuous HTS hit
compound, using structural information to guide design.
Transforming a nitrile moiety on the scaffold into a primary
amine led to a serendipitous and large increase in cell activity.
Optimization of in vitro ADMET properties was achieved by
incorporating an oxetane motif adjacent to a primary amine.
Efficacy of 22 in mouse EAE validates our mechanistic
hypothesis that modulation of PKCθ in vivo has potential for
the treatment of MS.
9, 184,
468
3.3
>10
8, 542,
286
0.45
0.21
2.5
>10
b
59, 86
>10
>10
ASSOCIATED CONTENT
Supporting Information
6, 392,
1020
77,
100
The Supporting Information is available free of charge on the
ACS Publications website.
aEnzyme Ki are expressed in nM. Cell IC50 are expressed in
Molecular formula strings (CSV)
μM. c% remaining @ 30 min; dIC50 and expressed in μM.
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theta-deficient mice. J. Immunol. 2006, 176,
2872-2879.
Chemistry and synthetic schemes, synthetic procedures and
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3
4
5
6
7
8
compound characterization, description of biochemical assays,
description of cellular assays, molecular modeling, EAE model,
physiochemical properties and DMPK assays (PDF)
(9) Salek-Ardakani, S,; So, T.; Halteman, B.
S.; Altman, A.; Croft, M. Protein kinase C휃
controls Th1 cells in experimental autoimmune
encephalomyelitis. J. Immunol. 2005, 175, 7635-
7641.
AUTHOR INFORMATION
Corresponding Author
(10) Marsland, B.J.; Soos, T.J.; Spath, G.;
Littman, D.R.; Kopf, M. Protein kinase Cθ is
*Email: philip_collier@vrtx.com. Phone: +1 (617) 341-6921.
*Email: heather_twin@vrtx.com. Phone: + 44 (12) 35438826.
critical for the development of in vivo
T
helper (Th)2 cell but not Th1 cell responses.
J. Exp. Med. 2004, 200, 181–189.
9
Author Contributions
(11) Giannoni, F.; Lyon, A.B.; Wareing, M.D.;
Dias, P.B.; Sarawar, S.R. Protein kinase Cθ is
not essential for T-cell-mediated clearance of
murine gamma herpes virus 68. J. Virol. 2005,
79, 6808–6813.
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All authors have given approval to the final version of the
manuscript.
ACKNOWLEDGMENT
(12) Marsland, B.J.; Nembrini, C.; Schmitz,
N.; Abel, B.; Krautwald, S.; Bachmann, M.F.;
Kopf, M. Innate signals compensate for the
The authors thank Dr. Barry Davis for assistance in collecting
some experimental data.
absence of PKC-θ during in vivo CD8+
T cell
ABBREVIATIONS
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Attur, M.; Hippen, K. L.; Brown, M.; Blazar, B.
R.; Abramson, S. B.; Lafaille, J. J.; Dustin,
M. L. Protein kinase C-theta mediates negative
feedback on regulatory T cell function. Science
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Protein kinase C, an elusive therapeutic
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Dixit, A.; Silakari, O. Protein kinase C-theta
ADMET, absorption, distribution, metabolism, excretion and
toxicity; HFL-1, human fibroblast cell line-1; HTS, high
throughput screening; IL-2, interleukin-2.
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