Design of Potent and Selective Covalent Inhibitors of Bruton's Tyrosine Kinase Targeting an Inactive Conformation

Article abstract of DOI:10.1021/acsmedchemlett.9b00317

Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK

Full text of DOI:10.1021/acsmedchemlett.9b00317

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Letter
Design of Potent and Selective Covalent Inhibitors of
Bruton’s Tyrosine Kinase Targeting an Inactive Conformation
Robert Pulz, Daniela Angst, Janet Dawson, Francois Gessier, Sascha Gutmann,
Rene Hersperger, Alexandra Hinniger, Philipp Janser, Guido Koch, Laszlo
Revesz, Anna Vulpetti, Rudolf Waelchli, Alfred Gilbert Zimmerlin, and Bruno Cenni
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00317 • Publication Date (Web): 06 Sep 2019
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Design of Potent and Selective Covalent Inhibitors of Bruton’s
Tyrosine Kinase Targeting an Inactive Conformation
Robert Pulza*, Daniela Angsta, Janet Dawsonb, Francois Gessiera, Sascha Gutmannc, Rene Herspergera,
Alexandra Hinnigerc, Philipp Jansera, Guido Kocha, Laszlo Revesza, Anna Vulpettia, Rudolf Waelchlia,
Alfred Zimmerlind, and Bruno Cennib
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aGlobal Discovery Chemistry, bAutoimmunity, Transplantation & Inflammation, cChemical Biology & Therapeutics, dPK
Sciences, Novartis Institutes for BioMedical Research, Novartis Campus, CH-4002 Basel, Switzerland
KEYWORDS BTK, covalent inhibitor, irreversible inhibitor, kinase inhibitor, kinase selectivity.
ABSTRACT: Bruton’s tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of
immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and
macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells
and immune-complex driven inflammation. We report the design, optimization and characterization of potent and selective covalent
BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent
irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and
showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this
compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in
compounds like 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.
Bruton’s tyrosine kinase (BTK) is a cytoplasmic tyrosine
kinase and a member of the TEC kinase family consisting of
BTK, tyrosine kinase expressed in hepatocellular carcinoma
(TEC), bone marrow X-linked tyrosine kinase (BMX),
interleukin-2-inducible T cell kinase (ITK) and tyrosine-
protein kinase TXK (TXK).1 BTK is selectively expressed in a
subset of immune cells, including macrophages, mast cells,
basophils, platelets and B cells, but not in plasma cells or T
cells.2,3 BTK contains an N-terminal pleckstrin homology (PH)
domain, a Src homology 3 (SH3) domain, a Src homology 2
domain and a C-terminal kinase domain. Its function is
regulated by membrane recruitment via the PH domain,
phosphorylation of Tyr551 in the activation loop and
autophosphorylation of Tyr223 in the SH3 domain.4 BTK is a
key regulator of antigen receptor signaling in B cells and of
Fcγ and Fcε receptor signaling in macrophages and mast cells,
respectively. Mutations in the BTK gene leading to BTK
deficiency or non-functional BTK protein are the most
frequent cause of X-linked agammaglobulinemia (XLA), a
human primary immunodeficiency characterized by the
absence of BTK protein and incomplete B cell development
caused by autoreactive B cells or immune-complex driven
inflammation. In addition, BTK inhibitors have been shown to
be clinically efficacious in various B cell malignancies.9
Over the last years, many pharmaceutical companies and
academic groups embarked on the identification and
development of BTK inhibitors, both irreversible and
reversible.10 Ibrutinib (1) (Figure 1) is approved for several B
cell malignancies including chronic lymphocytic leukemia and
irreversibly inhibits BTK through covalent modification of the
non-catalytic Cys481 residue in the ATP (adenosine
triphosphate) binding site of the kinase domain.11 Due to its
non-selective type I binding mode, 1 not only potently inhibits
BTK, but irreversibly inhibits all 10 kinases which carry a Cys
at the same position as BTK (Supporting information).11–13 In
addition, 1 inhibits several kinases noncovalently at clinically
relevant concentrations.12,14 Most importantly, 1 covalently
inhibits epidermal growth factor receptor kinase (EGFR)
kinase, which is associated with clinical adverse effects of skin
rash and diarrhea.15 The inhibition of TEC and proto-oncogene
tyrosine-protein kinase Src (SRC) is being discussed in the
context of clinical bleeding events with 1.14,16,17 Second
generation clinical-stage covalent inhibitors like spebrutinib
and acalabrutinib keep a similar binding mode to BTK as 1
and therefore, despite offering an overall improved kinase
selectivity profile, still inhibit several Cys-containing
kinases.13,18
resulting in the absence of mature
B
cells and
immunoglobulins.5 BTK deficient mice or mice carrying the
hypomorphic xid mutation in the BTK PH domain exhibit
defects in B cell development resulting in a phenotype similar
to human XLA, but with reduced severity.6 Pharmacological
intervention with BTK inhibitors has shown efficacy in animal
models of rheumatoid arthritis and systemic lupus
erythematosus.7,8 Based on this strong genetic and
pharmacological validation, it is likely that a BTK inhibitor
will have a positive impact on autoimmune diseases which are
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O
O
N
N
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H
N
HN
NH2
N
O
N
N
N
O
N
N
O
O
1
2
(CGI1746)
(Ibrutinib)
9
Figure 1. Selected BTK inhibitors
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Reversible BTK inhibitors with high kinase selectivity have
been described. CGI1746 (2) (Figure 1) was the first
compound to be reported to bind to a specific inactive
conformation of BTK which resulted in an outstanding kinase
selectivity profile, superior to the clinical irreversible
inhibitors.19
Figure 3. Co-crystal structure of the human BTK kinase domain
in complex with 3 (PDP 6S90). Potential attachment points
towards Cys481 (cyan) are shown with red arrows.
Based on this design concept we generated our first
prototype (4) with an acrylamide warhead attached to the
pyrrolopyrimidine via a methylene linker (Figure 2). In a BTK
enzymatic assay 4 exhibited an IC50 of 16 nM compared to 13
nM of the reversible starting point 3. The propionamide
derivative 5, which is devoid of an electrophilic warhead,
inhibited BTK only very weakly with an IC50 of 3.9 µM,
suggesting an important contribution of the acrylamide
warhead to the potency of 4.
While reversible inhibitors require continuous exposure of
the drug over the whole dosing interval in order to maintain a
high degree of target inhibition, the pharmacodynamic (PD)
effect of irreversible inhibitors can be decoupled from their
pharmacokinetics (PK). A short systemic exposure could be
sufficient for a sustained PD effect, since the duration of the
PD effect depends on the turnover of the target-inhibitor
adduct, rather than the duration of exposure of the inhibitor. In
addition, irreversible inhibitors offer higher potency and
therefore potentially lower human doses.20
Additional evidence for a covalent mode of action was
generated by incubation of 4 with the human BTK kinase
domain protein, revealing a single peak mass corresponding to
the 1:1 adduct of BTK and 4 in LC/MS analysis (Supporting
information). Furthermore, after preincubation of full-length
human BTK and 4 at high concentrations, the enzymatic
activity was not rescued by dilution (Supporting information).
This indicates an irreversible mode of action for 4, similar to
rac-1 and in contrast to the reversible inhibitor 5.
Hence, during the course of our BTK inhibitor program the
strategy was selected to combine the highly specific binding
mode of CGI1746-like reversible inhibitors with the potency
and PK/PD advantage of the irreversible mode of action. As
starting point we used the selective reversible inhibitor 3
(Figure 2, see Supporting information for selectivity data), a
combination of an internal pyrrolopyrimidine screening hit
with the tail fragment of 2.21
We next assessed whether 4 maintains the binding mode of
3 to the inactive BTK conformation. In Ramos cells, 4
inhibited phosphorylation of Tyr551 over time in contrast to
the results obtained for rac-1 (Figure 4). This is in line with
the expected sequestration of Tyr551 as previously reported
for 2.19 In addition, this suggests that the cellular turn-over of
Tyr551 phosphorylation is very rapid and allows for fast
binding and inhibition of cellular BTK in presence of pathway
activation.
N
N
N
N
H
H
N
N
HN
HN
O
O
N
R
3
O
N
O
O
4
5
N
300
Figure 2. Reversible starting point 3, covalent prototype 4 and its
reversible analog 5
rac-1
4
The co-crystal structure of 3 with the human BTK kinase
domain (Figure 3) revealed the stabilization of an inactive
BTK conformation similar to 2 leading to a sequestration of
Tyr551, which shields it from phosphorylation by the
upstream kinases in the pathway. Based on this co-crystal
structure, the pyrrolopyrimidine hinge binder and the phenyl
ring occupying the hydrophobic channel of the protein are in
close enough proximity to Cys481 to provide suitable exit
vectors for linkers containing electrophilic warheads. Since we
intended to keep our inhibitors as small as possible to allow
for good physico-chemical properties we decided to attach the
linker directly to the pyrrolopyrimidine while removing the
hydrophobic channel substituent.
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100
0
0
10
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Time [min]
30
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Figure 4. Time course of Tyr551 phosphorylation in Ramos cells
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8
in presence of 0.5 µM compound (in % of a maximally stimulated
control).
***
*
***
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80
60
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0
As a consequence of its specific binding mode, 4 exhibits an
excellent kinase selectivity profile. This is probaly due to a
higher dissimilarity of the kinases within the so called "H3"
pocket formed around Tyr551 compared to the ATP binding
site as well as the possible inability of kinases other than BTK
to adopt this specific conformation.19 In a panel of 61 non-Cys
containing kinases including SRC, 4 did not show any IC50
<
9
10 µM (Supporting information). Moreover, it also did not
inhibit the Cys-containing kinases EGFR, receptor tyrosine-
protein kinase erbB-2 (ERBB2), receptor tyrosine-protein
kinase erbB-4 (ERBB4), and Janus kinase 3 (JAK3) and
showed a 312-fold selectivity over BMX, the most related
kinase to BTK (Table 1 and Supporting information). The
promising biochemical selectivity profile could be confirmed
in cellular assays. While 4 potently blocked the BTK-
dependent FcγR-induced IL8 release in THP1 cells with an
IC50 of 0.08 µM, it did not inhibit EGFR phosphorylation in
A431 cells up to the highest concentration of 8 µM. In
contrast, 1 inhibited BMX, EGFR, and ERBB4 with similar
potency to BTK in biochemical assays, as well as cellular
pEGFR. Taken together, these data validate our design
concept of transforming a selective reversible scaffold into a
selective irreversible inhibitor.
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Ctrl
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20
4
60 mg/kg
rac-1
Figure 5. Dose dependent inhibition of reverse passive Arthus
reaction (RPA) in mice (FcγR-dependent skin swelling) 5 hours
after a single p.o. dose of 4 compared to rac-1 and vehicle control
(error bars indicate SD). * p ≤ 0.05, *** p ≤ 0.001.
With our prototype 4 we had identified a selective covalent
BTK inhibitor with in vivo efficacy after oral dosing.
Nevertheless, several properties of
4 required further
optimization. First, the cellular potency with an IC50 of 0.934
µM in human blood was significantly weaker than for 1 (IC50
of 0.066 µM) (Table 1). Second, the ADME properties of 4
were suboptimal, as the compound was highly unstable in
mouse, rat, and human microsomes with intrinsic clearance
values of 554, 693, and >700 µL min-1 mg-1, respectively.
Although covalent irreversible inhibitors do not require
continuous exposure, the fast elimination of 4 resulted in
rather high doses for efficacy in the mouse RPA. The PK
profile was even poorer in rat, where 4 showed an extremely
high clearance of >300 mL min-1 kg-1 resulting in a very low
oral bioavailability (BAV) of 2 % (Supporting information).
Table 1. Potency and selectivity data for 1 and 4
IC50 [µM]a
BTKb
BMXb
EGFRb
1
4
0.014 (0.016)
5.0 (0)
0.005f
0.001 (0)
0.016f
> 10
ERBB4b
0.007f
> 10f
Cellular FcγR/IL8c
Cellular pEGFR/A431d
Human blood CD69e
< 0.005
0.080 (0.0007)
> 8f
0.045 (0.015)
0.066 (0.022)
Although the in vivo clearance was too high to be solely
driven by hepatic metabolism, we aimed to increase the
stability in liver microsomes first. An in vitro metabolite
identification study after incubation with rat liver microsomes
(RLM) revealed the product of an oxidation of the t-butyl
group as the most prominent metabolite. Therefore, we
replaced it with a more stable cyclopropyl group in 6, which
did not affect stability in RLM, but interestingly slightly
reduced human microsomal clearance (Table 2). The loss of
potency of 6 could be compensated by the introduction of a
fluorine at the central aromatic ring (7), which could be due to
a multipolar interaction of the fluorine with the carbonyl group
of Gly409 of the P-loop.23
0.934 (0.407)
a) IC50 determined as a mean (n ≥ 2) (SD); b) enzyme assay; c)
FcγR-induced IL8 release in THP1 cells; d) EGFR
phosphorylation in A431 cells; e) anti-IgM/IL-4 induced CD69
expression on B cells in human blood; f) single experiment.
Since our data demonstrated that 4 blocks cellular FcγR-
dependent response, we tested its effect on FcγR-mediated
hypersensitivity in vivo. For that, we chose the mouse reverse
passive Arthus reaction (RPA), where an acute
hypersensitivity reaction is induced by intravenous injection of
ovalbumin (OVA) antigen, followed by intradermal injection
of a polyclonal Immunoglobulin G (IgG) anti-OVA antibody.
For 1, efficacy in the mouse RPA has been described
previously.22 We used rac-1 as a positive control, which
exhibits very similar in vitro and in vivo properties compared
to the (R)-enantiomer 1. Treatment with a single oral dose of
4, given 2 hours prior to the induction of the RPA response,
dose dependently reduced skin swelling measured 5 hours
after dosing the compound. Maximal inhibition of 65 % was
seen with the 60 mg/kg dose (Figure 5). In comparison, rac-1
reached 84 % inhibition of swelling with a 30 mg/kg dose.
Terminal blood concentrations of 4 were 4, 14 and 118 nM for
the 6, 20, and 60 mg/kg dose, respectively (compared to 187
nM for rac-1.
We then focused our attention on the linker between the
pyrrolopyrimidine hinge binder and the acryl amide warhead.
We hypothesized that by modification of the linker we could
not only increase metabolic stability but also increase potency
of the compounds by providing a more optimal orientation of
the warhead towards Cys481, allowing for a faster formation
of the covalent bond. We quickly realized that by optimizing
the compounds we hit the limits of the biochemical BTK
inhibition assay which was in the range of an IC50 of 1-2 nM
(half of the enzyme concentration present in the assay).
Therefore our optimization of these potent compounds was
mostly led by the more relevant and discriminating human
blood B cell inhibition assay. Extension of the linker by one
carbonyl group (8) provided a compound with highly
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improved biochemical and cellular potency, in addition to a
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1
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3
4
5
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8
0.006
(0.002)
0.066
(0.004)
good stability in liver microsomes. The hydroxy-azetidine 9
further improved microsomal stability, but lost nearly 20 fold
in human blood potency vs 8. Increasing the ring size to a 3-
pyrrolidine (10) did not affect in vitro potency, while the 3-
piperidine (11) clearly provided a less suitable positioning as
indicated by a ~300 fold loss in biochemical potency
compared to 9 and 10. Both, the 4-piperidine (12) and the 4-
tetrahydropyridine (13) linker showed similar biochemical
potency as 9 and 10, but these compounds were significantly
more potent in the human blood assay with IC50 values of 19
and 32 nM, respectively. The high potency of compounds like
13 allowed the introduction of substituted, less-reactive
warheads. Both, the aminomethyl-substituted acrylamide 14
and the but-2-enamide 15 showed a remarkably high potency
in the human blood assay with IC50 values of 26 and 66 nM,
respectively. Unfortunately, none of the compounds 10-15
showed a good stability in rat or human liver microsomes.
15
127 / 459
N
O
a) BTK enzyme inhibition IC50 mean (SD) (n ≥ 2); b) anti-
IgM/IL-4 induced CD69 expression in human blood B cell IC50
mean (SD) (n ≥ 2); c) intrinsic clearance in rat and human liver
microsomes [L∙min-1∙mg-1 protein], n = 1; d) Single
experiment; e) mean of two experiments.
As expected from the in vitro microsomal stability, 13, 14,
9
and 15 showed fast elimination after intravenous dosing in rat
PK studies with clearance values of >300, 176, and >300 mL
min-1 kg-1, respectively. Unexpectedly, even 8 (300 mL min-1
kg-1) and 9 (183 mL min-1 kg-1) did not show improved in vivo
clearance despite good stability in rat liver microsomes
(Supporting information). Since the clearance values were
considerably above the hepatic blood flow of the rats (50-100
mL min-1 kg-1), it is likely that extrahepatic pathways
contributed significantly to the fast elimination. Additional in
vitro profiling did not provide indications for the extrahepatic
clearance mechanism, since the compounds generally neither
showed significant instability in plasma nor in liver S9
fractions.
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Table 2. SAR for covalent pyrrolopyrimidines
N
N
H
N
R2 = H
R2 = F
6
7 15
-
HN
O
R1
R2
Despite not being able to significantly improve the rat
clearance compared to the prototype 4, the compounds were
considerably more potent in the in vitro human blood CD69
assay. To assess a potential progress in in vivo efficacy of the
compounds compared to 4 we measured BTK occupancy
(Supporting information), which has been shown to correlate
with pathway inhibition in several BTK-dependent animal
models.24 We assessed BTK occupancy in spleen homogenates
of female rats 5 h after an oral dose of 10 mg kg-1 of the
WB
CD69
IC50
BTK
IC50
RLM /
HLM
R1
[µM]a
CLintc
[µM]b
N
6
7
0.190d
0.026d
7.47d
578 / 252
O
compounds (Figure 6). Compared to
a mean splenic
1.26
(0.52)
>700 /
308
N
occupancy of 93% for rac-1, no significant BTK occupancy
could be measured for 4. This was in line with the compound
concentrations in blood we detected 0.5, 2, and 5 h after
dosing. The blood levels for rac-1 were 67, 82, and 15 nM,
whereas no exposure could be measured for 4. Amongst other
factors like sub-optimal permeability and solubility, the
exposure of 4 in rat might be limited by a significant first-pass
metabolism. Despite very low blood levels (4.2, 2.2, and 1.5
nM), 8 showed occupancy of 44%. In a separate experiment,
14 showed no significant BTK occupancy, whereas 15
provided 70% (blood levels 14 and 15 at 5 h were 3 and 16
nM, respectively). Finally, 13 achieved the same BTK
occupancy as rac-1 (96%), despite significantly lower blood
levels at 5 h (6 nM for 13, 86 nM for rac-1).
O
O
0.0012
(0.0007)
0.016
(0.001)
8
9
98e / 39e
N
O
OH
0.0014
(0.0004)
0.303
(0.226)
41 / 3
N
O
O
0.005
(0.003)
0.225
(0.095)
10
11
171 / 111
N
N
O
0.390
(0.434)
8.00
(2.83)
>700 /
603
0.001
(0.0003)
0.019
(0.004)
12
13
14
578 / 126
408 / 660
N
N
O
O
0.0009
(0.0005)
0.032
(0.021)
0.0006
(0.0002)
0.026
(0.006)
628 /
>700
N
N
O
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D. Eichlisberger, S. Gaveriaux, and A.-S. Mangold for their
100
75
50
25
0
1
2
3
4
5
6
7
efforts towards developing and performing biochemical and
cellular assays, P. Ramseier and M. Vogelsanger for in vivo
pharmacology and G. Weckbecker for scientific guidance. We are
grateful to the MX beamline team at the Swiss Light Source (PSI
Villigen, Switzer-land) for outstanding support at the beamline
and Expose GmbH (Switzerland) for diffraction data collection.
ABBREVIATIONS
8
9
ADME, absorption, distribution, metabolism, and excretion; ATP,
adenosine triphosphate; BAV, bioavailability; BCR, B-cell
receptor; BMX, bone marrow X-linked tyrosine kinase; BTK,
Bruton’s tyrosine kinase; CD69, cluster of differentiation 69;
EGFR, epidermal growth factor receptor; ERBB2, receptor
tyrosine-protein kinase erbB-2; ERBB4, receptor tyrosine-protein
kinase erbB-4; ITK, interleukin-2-inducible T cell kinase; IgG,
Immunoglobulin G; JAK3, Janus kinase 3; OVA, ovalbumin;
PAMPA, parallel artificial membrane permeability assay; PH,
-25
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//
1
4
8
1
-
-
13
14
15
rac
rac
Vehicle
Vehicle
Figure 6. BTK occupancy in rat spleen homogenate 5 h after a 10
mg kg-1 p.o. dose of compounds compared to the respective
vehicle control. Circles represent single animal values, lines
indicate average ± SD.
pleckstrin
homology;
PK/PD,
pharmacokinetic/pharmacodynamic; RLM, rat liver microsomes;
RPA, reverse passive Arthus reaction; SH3, Src homology 3;
SRC, proto-oncogene tyrosine-protein kinase Src; TEC, tyrosine-
protein kinase Tec; TXK, tyrosine-protein kinase TXK;
Finally, we confirmed the kinase selectivity of our
optimized inhibitors by testing 8 in a biochemical kinase panel
containing 60 kinases, including Cys-containing kinases
BMX, EGFR, ERBB2 and JAK3 (Supporting information). 8
showed an excellent profile providing a 40-fold selectivity
over BMX (BMX IC50 49 nM) and a >25’000 fold selectivity
for all other kinases tested.
REFERENCES
(1)
(2)
(3)
(4)
(5)
Robinson, D. R.; Wu, Y.-M.; Lin, S.-F. The Protein Tyrosine
Kinase Family of the Human Genome. Oncogene 2000, 19,
5548–5557.
Satterthwaite, A. B. Independent and Opposing Roles For Btk
and Lyn in B and Myeloid Signaling Pathways. J. Exp. Med.
1998, 188, 833–844.
Koprulu, A. D.; Ellmeier, W. The Role of Tec Family Kinases
in Mononuclear Phagocytes. Crit. Rev. Immunol. 2009, 29, 317–
333.
Mano, H. Tec Family of Protein-Tyrosine Kinases: An
Overview of Their Structure and Function. Cytokine Growth
Factor Rev. 1999, 10, 267–280.
In summary, we converted pyrrolopyrimidine-based
selective reversible inhibitors into a series of potent and
selective irreversible BTK inhibitors with in vivo activity.
Similar to what has in the meantime been published by
others,25 the combination of binding to an inactive
conformation of BTK with the covalent irreversible MoA
resulted in compounds with a high kinase selectivity even over
several closely-related Cys-containing kinases. In addition,
these compounds provided high BTK occupancy after oral
dosing, similar to 1. Due to the very high clearance of these
compounds, we decided to abandon this series in favor of an
alternative scaffold. This resulted in the discovery of a highly
potent and selective clinical candidate, which will be
described in due course.
Conley, M. E.; Dobbs, A. K.; Farmer, D. M.; Kilic, S.; Paris, K.;
Grigoriadou, S.; Coustan-Smith, E.; Howard, V.; Campana, D.
Primary
B
Cell Immunodeficiencies: Comparisons and
Contrasts. Annu. Rev. Immunol. 2009, 27, 199–227.
(6)
(7)
Mestas, J.; Hughes, C. C. W. Of Mice and Not Men: Differences
between Mouse and Human Immunology. J. Immunol. 2004,
172, 2731–2738.
Xu, D.; Kim, Y.; Postelnek, J.; Vu, M. D.; Hu, D.-Q.; Liao, C.;
Bradshaw, M.; Hsu, J.; Zhang, J.; Pashine, A.; Srinivasan, D.;
Woods, J.; Levin, A.; O’Mahony, A.; Owens, T. D.; Lou, Y.;
Hill, R. J.; Narula, S.; DeMartino, J.; Fine, J. S. RN486, a
Selective Bruton’s Tyrosine Kinase Inhibitor, Abrogates
Immune Hypersensitivity Responses and Arthritis in Rodents. J.
Pharmacol. Exp. Ther. 2012, 341, 90–103.
ASSOCIATED CONTENT
Supporting Information
Full descriptions of all biological assays and in vivo studies.
Kinase selectivity data. Crystallographic data collection and
refinement statistics for crystal structures. Experimental
procedures and characterization of compounds. The Supporting
Information is available free of charge on the ACS Publications
website.
(8)
Satterthwaite, A. B. Bruton’s Tyrosine Kinase, a Component of
B
Cell Signaling Pathways, Has Multiple Roles in the
Pathogenesis of Lupus. Front. Immunol. 2018, 8, 1–10.
Aw, A.; Brown, J. R. Current Status of Bruton’s Tyrosine
Kinase Inhibitor Development and Use in B-Cell Malignancies.
Drugs Aging 2017, 34, 509–527.
(9)
(10)
Zhang, Z.; Zhang, D.; Liu, Y.; Yang, D.; Ran, F.; Wang, M. L.;
Zhao, G. Targeting Bruton’s Tyrosine Kinase for the Treatment
of B Cell Associated Malignancies and Autoimmune Diseases:
Preclinical and Clinical Developments of Small Molecule
Inhibitors. Arch. Pharm. (Weinheim). 2018, 351, 1700369.
Honigberg, L. A.; Smith, A. M.; Sirisawad, M.; Verner, E.;
Loury, D.; Chang, B.; Li, S.; Pan, Z.; Thamm, D. H.; Miller, R.
A.; Buggy, J. J. The Bruton Tyrosine Kinase Inhibitor PCI-
32765 Blocks B-Cell Activation and Is Efficacious in Models of
Autoimmune Disease and B-Cell Malignancy. Proc. Natl. Acad.
Sci. 2010, 107, 13075–13080.
AUTHOR INFORMATION
Corresponding Author
* Tel: +41 79 310 6846. E-mail: robert.pulz@novartis.com
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Médard, G.; Pachl, F.; Ruprecht, B.; Klaeger, S.; Heinzlmeir, S.;
Helm, D.; Qiao, H.; Ku, X.; Wilhelm, M.; Kuehne, T.; Wu, Z.;
Dittmann, A.; Hopf, C.; Kramer, K.; Kuster, B. Optimized
We thank E. Blum, O. Decoret, F. Gruber, J. Karrer, M.-L.
Manisse, W. Miltz, B. Niepold, F. Ossola, M. Regenscheit, G.
Rose, and S. Wildpreth for their synthetic support, P. Drueckes,
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Chemical Proteomics Assay for Kinase Inhibitor Profiling. J.
Proteome Res. 2015, 14, 1574–1586.
1
2
3
4
5
6
7
8
(13)
(14)
Barf, T.; Covey, T.; Izumi, R.; van de Kar, B.; Gulrajani, M.;
van Lith, B.; van Hoek, M.; de Zwart, E.; Mittag, D.; Demont,
D.; Verkaik, S.; Krantz, F.; Pearson, P. G.; Ulrich, R.; Kaptein,
A. Acalabrutinib (ACP-196): A Covalent Bruton Tyrosine
Kinase Inhibitor with a Differentiated Selectivity and In Vivo
Potency Profile. J. Pharmacol. Exp. Ther. 2017, 363, 240–252.
Nicolson, P. L. R.; Hughes, C. E.; Watson, S.; Nock, S. H.;
Hardy, A. T.; Watson, C. N.; Montague, S. J.; Clifford, H.;
Huissoon, A. P.; Malcor, J.-D.; Thomas, M. R.; Pollitt, A. Y.;
Tomlinson, M. G.; Pratt, G.; Watson, S. P. Inhibition of Btk by
Btk-Specific Concentrations of Ibrutinib and Acalabrutinib
Delays but Does Not Block Platelet Aggregation Mediated by
Glycoprotein VI. Haematologica 2018, 103, 2097–2108.
Kaur, V.; Swami, A. Ibrutinib in CLL: A Focus on Adverse
Events, Resistance, and Novel Approaches beyond Ibrutinib.
Ann. Hematol. 2017, 96, 1175–1184.
Shatzel, J. J.; Olson, S. R.; Tao, D. L.; McCarty, O. J. T.;
Danilov, A. V.; DeLoughery, T. G. Ibrutinib-Associated
Bleeding: Pathogenesis, Management, and Risk Reduction
Strategies. J. Thromb. Haemost. 2017, 38, 1–13.
Atkinson, B. T.; Ellmeier, W.; Watson, S. P. Tec Regulates
Platelet Activation by GPVI in the Absence of Btk. Blood 2003,
102, 3592–3599.
Evans, E. K.; Tester, R.; Aslanian, S.; Karp, R.; Sheets, M.;
Labenski, M. T.; Witowski, S. R.; Lounsbury, H.; Chaturvedi,
P.; Mazdiyasni, H.; Zhu, Z.; Nacht, M.; Freed, M. I.; Petter, R.
C.; Dubrovskiy, A.; Singh, J.; Westlin, W. F. Inhibition of Btk
with CC-292 Provides Early Pharmacodynamic Assessment of
Activity in Mice and Humans. J. Pharmacol. Exp. Ther. 2013,
346, 219–228.
Di Paolo, J. A.; Huang, T.; Balazs, M.; Barbosa, J.; Barck, K.
H.; Bravo, B. J.; Carano, R. A. D. D.; Darrow, J.; Davies, D. R.;
DeForge, L. E.; Diehl, L.; Ferrando, R.; Gallion, S. L.;
Giannetti, A. M.; Gribling, P.; Hurez, V.; Hymowitz, S. G.;
Jones, R.; Kropf, J. E.; Lee, W. P.; Maciejewski, P. M.;
Mitchell, S. A.; Rong, H.; Staker, B. L.; Whitney, J. A.; Yeh, S.;
Young, W. B.; Yu, C.; Zhang, J.; Reif, K.; Currie, K. S. Specific
Btk Inhibition Suppresses B Cell– and Myeloid Cell–Mediated
Arthritis. Nat. Chem. Biol. 2011, 7, 41–50.
9
10
11
12
13
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15
16
17
18
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24
25
26
27
28
29
30
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43
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45
46
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48
49
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51
52
53
54
55
56
57
58
59
60
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The
Resurgence of Covalent Drugs. Nat. Rev. Drug Discov. 2011,
10, 307–317.
Thakkar, M.; Koul, S.; Bhuniya, D.; Singh, U. Preparation of
Substituted Heterobicyclic Compounds, Compositions and
Medicinal Applications Thereof. WO2013/157021 A1, 2013.
Chang, B. Y.; Huang, M. M.; Francesco, M.; Chen, J.;
Sokolove, J.; Magadala, P.; Robinson, W. H.; Buggy, J. J. The
Bruton Tyrosine Kinase Inhibitor PCI-32765 Ameliorates
Autoimmune Arthritis by Inhibition of Multiple Effector Cells.
Arthritis Res. Ther. 2011, 13, R115.
Paulini, R.; Müller, K.; Diederich, F. Orthogonal Multipolar
Interactions in Structural Chemistry and Biology. Angew.
Chemie Int. Ed. 2005, 44, 1788–1805.
Goess, C.; Harris, C. M.; Murdock, S.; McCarthy, R. W.;
Sampson, E.; Twomey, R.; Mathieu, S.; Mario, R.; Perham, M.;
Goedken, E. R.; Long, A. J. ABBV-105, a Selective and
Irreversible Inhibitor of Bruton’s Tyrosine Kinase, Is
Efficacious in Multiple Preclinical Models of Inflammation.
Mod. Rheumatol. 2019, 29, 510–522.
(23)
(24)
(25)
Liang, Q.; Chen, Y.; Yu, K.; Chen, C.; Zhang, S.; Wang, A.;
Wang, W.; Wu, H.; Liu, X.; Wang, B.; Wang, L.; Hu, Z.; Wang,
W.; Ren, T.; Zhang, S.; Liu, Q.; Yun, C.-H.; Liu, J. Discovery of
N-(3-(5-((3-Acrylamido-4-(morpholine-4-
carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-
yl)-2-methylphenyl)-4-(tert-butyl)benzamide
(CHMFL-BTK-
01) as a Highly Selective Irreversible Bruton’s Tyrosine Kinase
(BTK) Inhibitor. Eur. J. Med. Chem. 2017, 131, 107–125.
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Selective covalent
BTK inhibitor (8)
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