Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase

Article abstract of DOI:10.1021/acs.jmedchem.9b01916

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC₉₀ of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.

Full text of DOI:10.1021/acs.jmedchem.9b01916

Subscriber access provided by La Trobe University Library
Article
Discovery of LOU064 (Remibrutinib), a Potent and Highly
Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase
Daniela Angst, François Gessier, Philipp Janser, Anna Vulpetti, Rudolf Wälchli,
Christian Beerli, Amanda Littlewood-Evans, Janet Dawson, Barbara Nuesslein-
Hildesheim, Grazyna Wieczorek, Sascha Gutmann, Clemens Scheufler, Alexandra
Hinniger, Alfred Gilbert Zimmerlin, Enrico G. Funhoff, Robert Pulz, and Bruno Cenni
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01916 • Publication Date (Web): 21 Feb 2020
Downloaded from pubs.acs.org on February 22, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of LOU064 (Remibrutinib), a Potent and
Highly Selective Covalent Inhibitor of Bruton’s
Tyrosine Kinase
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
a
a
a
Daniela Angst* , François Gessier , Philipp Janser , Anna Vulpetti , Rudolf Wälchli , Christian
b
b
b
b
Beerli , Amanda Littlewood-Evans , Janet Dawson , Barbara Nuesslein-Hildesheim , Grazyna
b
c
c
c
d
Wieczorek , Sascha Gutmann , Clemens Scheufler , Alexandra Hinniger , Alfred Zimmerlin ,
e
a
b
Enrico G. Funhoff , Robert Pulz , Bruno Cenni
a
b
c
Global Discovery Chemistry, Autoimmunity, Transplantation & Inflammation, Chemical
d
e
Biology & Therapeutics, PK Sciences, Preclinical Safety, Novartis Institutes for BioMedical
Research, Novartis Campus, CH-4002 Basel, Switzerland
Abstract. Bruton’s tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in
immunity and is considered an attractive target for treating autoimmune diseases. The use of
currently marketed covalent BTK inhibitors is limited to oncology indications based on their
suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064
(remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an
exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential
for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
demonstrates potent in vivo target occupancy with an EC of 1.6 mg/kg and dose-dependent
9
0
efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in Phase 2 clinical
studies for chronic spontaneous urticaria and Sjoegren’s Syndrome.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Bruton’s tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase selectively expressed in a subset
of immune cells, including macrophages, mast cells, basophils, platelets and B cells, but not in
1
,2
mature plasma cells and T cells. BTK is activated by membrane recruitment via its pleckstrin
homology domain and phosphorylation of the activation loop Tyr551 by SYK and SRC family
1,2
kinases, resulting in autophosphorylation of Tyr223 in the SH3 domain. BTK is an essential
node and rheostat for the signaling of the antigen receptor in B cells (BCR) and of activating Fc
receptors for IgG and IgE (FcγR, FcεR) in macrophages and mast cells.3–5 While inborn BTK
deficiency does not affect the development of many immune cells, it results in severely impaired
B cell development and antibody production.6–8 In humans BTK deficiency is the most frequent
cause of the primary immunodeficiency X-linked agammaglobulinemia (XLA), a condition that
9
if untreated is associated with an increased bacterial infection risk. Today XLA is treated by
immunoglobulin replacement therapy suggesting that the main risk factor for infections is the
absence of protective immunoglobulins rather than the deficiency in cellular BTK function.10
The role of BTK appears not to be critical for B cell survival once the B cells have developed,
suggesting that BTK inhibition after infancy may not lead to acute B cell and antibody
depletion.6
ACS Paragon Plus Environment
2

Page 3 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The potential role of BTK in autoimmunity, allergy and chronic inflammatory conditions is
supported by genetic validation in many rodent models for arthritis, systemic lupus
erythematosus and anaphylaxis.3,11–13 More recently, the functional relevance of BTK has also
been validated by pharmacological inhibition in various clinical trials and is testament for the
activities across many pharmaceutical companies to identify and develop BTK inhibitors, both
covalent and reversible.14–22
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The kinase domain of BTK contains Cys481 in a position conserved in a group of eleven kinases
including the closely related TEC family kinases BMX, TEC, ITK and TXK, as well as in
EGFR, ERBB2, ERBB4, JAK3, BLK and MKK7.23,24 These conserved cysteines have been
targeted in the development of covalent inhibitors of BTK, EGFR, JAK3 and MKK7. The
concept of selective targeted covalent kinase inhibitors has received more attention due its
potential for sustained target inhibition, uncoupled from sustained and high systemic drug
exposure. This special pharmacologic feature of covalent inhibitors may lead to high efficacy
and an improved safety profile.25,26
The covalent BTK inhibitors ibrutinib (1) and acalabrutinib (2) (Figure 1, a)) have shown
efficacy and an attractive risk-benefit profile leading to their approval in several B cell
2
7
malignancies including chronic lymphocytic leukemia and mantle cell lymphoma. Due to its
binding to BTK in its active conformation, ibrutinib (1) not only potently inhibits BTK, but
irreversibly inhibits all kinases which carry a Cys at the same position as BTK, and in addition
inhibits reversibly several other kinases.14,28,29 While the clinical efficacy of 1 is of significant
benefit in difficult to treat oncologic indications, its side effect profile precludes its use in
rheumatologic indications.30–34 It is likely that its off-target activity is associated with the clinical
3
5
risk for skin rash and diarrhea (well established for EGFR inhibitors ), or platelet disfunction
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
shown for TEC and SRC kinases36,37). Second generation clinical-stage covalent inhibitors such
as acalabrutinib (2), tirabrutinib (3), evobrutinib (4) and branebrutinib (5) (Figure 1, a)) retain a
similar binding mode to BTK as 1 and therefore, despite offering an overall improved kinase
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
selectivity profile, still inhibit several Cys-containing kinases.18,22,38–40
a)
N
O
O
O
N
NH
NH2
N
NH2
N
N
N
NH2
N
N
N
O
N
N
N
O
N
N
N
O
O
1
, ibrutinib
2, acalabrutinib
3, tirabrutinib
b)
F
O
O
NH2
H N
2
N
NH
N
O
HN
NH
N
N
O
H
O
N
N
O
N
O
4, evobrutinib
5, branebrutinib
N
H
O
6
, CGI-1746
Figure 1. a) Selected examples of covalent BTK inhibitors (1-5); b) Structure of CGI-1746 (6),
the first reported compound binding to an inactive BTK conformation.
Reversible BTK inhibitors with high kinase selectivity have been described15,16,21,41 and
CGI-1746 (6) (Figure 1, b)) was the first compound reported to bind to an inactive conformation
ACS Paragon Plus Environment
4

Page 5 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of BTK resulting in an excellent kinase selectivity. This binding mode prevents the
phosphorylation of Tyr551in the activation loop of BTK by upstream kinases and possibly
4
2
contributes to higher cellular potency. In contrast to covalent inhibitors, reversible inhibitors
require continuous systemic drug exposure over the entire dosing interval in order to maintain a
high degree of target inhibition. In order to achieve this, intensive efforts were necessary to
optimize the profile of 6, including ADME properties, potency, and safety, while maintaining the
specific binding mode, finally leading to GDC-0853 (fenebrutinib), a selective reversible BTK
inhibitor currently in Phase 2 clinical trials.21
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We were attracted by the concept of covalent BTK inhibition as it allows the optimization of
compounds for a potent and sustained pharmacodynamic effect without the need for extended
25
and high systemic compound exposure. At the same time, we wanted to differentiate from
competitor molecules by aiming for a very high selectivity to minimize kinase off-target
reversible and covalent binding. Therefore we selected the strategy of combining the advantages
of the covalent MoA with targeting the inactive conformation of BTK.
RESULTS AND DISCUSSION
The concept of combining a reversible BTK inhibitor binding to an inactive conformation of
BTK with a warhead leading to highly selective, covalent BTK inhibitors has been recently
published by us and others. 43,44 We disclosed the discovery of a series of pyrrolopyrimidines.
Using compound 7 (BTK enzyme IC of 0.013 ± 0.0078 M, Figure 2) as starting point, a
5
0
reversible BTK inhibitor binding to the same inactive BTK conformation as 6, we designed
inhibitors forming a covalent bond with Cys481. The compounds of this series, represented by 8
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
BTK enzyme IC of 0.0009 ± 0.0005 M, Figure 2), are potent inhibitors of BTK in vitro and
5
0
we were able to demonstrate BTK occupancy after oral dosing in a PK/PD model. However, the
pyrrolopyrimidine series suffered from very high in vivo clearance, making it challenging to
achieve in vivo efficacy at low doses. Thorough investigation of the pyrrolopyrimidine SAR did
not lead to a significantly improved PK profile. Therefore, we decided to explore alternative
hinge binders with the aim of improving the ADME properties, while keeping the key
interactions with BTK, as displayed in the X-ray cocrystal structure of 7 (Figure 3). Replacing
the pyrrolopyrimidine moiety by 6-aminopyrimidine as hinge binder motif provided 9 (Figure 2)
as a reversible prototype. Figure 3 shows the model of the aminopyrimidine 9 bound to the
inactive conformation of BTK based on the solved crystal structure with 7. In the model, the
aminopyrimidine scaffold forms the same key interactions with BTK as the pyrrolopyrimidine 7
with the exception of the contacts at the entrance of the ATP binding pocket. The removed
phenyl at position 6 of compound 7 is nicely sandwiched between the side chains of Leu408 and
Gly480. For that reason, some loss of affinity was expected.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
O
N
O
N
O
HN
HN
F
HN
F
N
4
O
N
5
5
6
N
H
N
H
N
N
N
NH2
7
N
8
9
BTK IC50 0.013 M
BTK IC50 0.0009 M
BTK IC50 7.3 M
Figure 2. Morphing of the pyrrolopyrimidine scaffold to an aminopyrimidine scaffold. BTK
enzyme inhibition in Caliper assay, IC shown.
50
ACS Paragon Plus Environment
6

Page 7 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Thr474
Lys430
Met477
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Tyr551
Cys481
Figure 3. Crystal structure of 7 (PDB 6S90; in cyan) in complex with human BTK overlaid with
the binding model of compound 9. H-bonds of the pyrrolopyrimidine and 6-aminopyrimidine
scaffold (in purple) to Met477 and a water molecule (red sphere) are highlighted (black dashed
lines), as well as the hydrogen bond between the carbonyl of the amide of the ligand and Lys430
side chain.
Indeed, compound 9 inhibited BTK in an enzymatic assay with an IC of 7.3 ± 0.35 M,
5
0
confirming our morphing idea. Similarily to what we described for the pyrrolopyrimidine
4
3
scaffold, we envisaged to generate potent and selective covalent inhibitors by introducing a
linker with a warhead at position 5 of the pyrimidine moiety to reach Cys481. We started with
acrylamide warheads, as they have been used successfully in covalent BTK inhibitors such as 1
and 4.45
SAR: Optimizing in vivo BTK Occupancy. We first investigated alkene based linkers
between the pyrimidine core and the warhead providing a somewhat rigidified exit vector.
Introducing a three carbon linker (10) and a four carbon linker (11) led to moderately potent
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compounds in the BCR/CD69 assay in blood (Table 1). However, 10 and 11 suffered from very
high clearance in rat liver microsomes (CL 924 and 815 L/min/mg, respectively). Replacing
int
the carbon linkers with 3-oxypyrrolidine (12) was not tolerated with respect to BTK inhibition,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
but the metabolic stability significantly increased (CL 135 L/min/mg). Switching to a
int
3
-oxyazetidine linker (13) led to an improvement of blood BCR/CD69 potency and good
metabolic stability (CL 25 L/min/mg). Having succesfully combined these two properties, we
int
sought to assess the in vivo PD effect of 13 by measuring BTK occupancy in rat spleen after a
single oral dose (Experimental Section). Administration of 13 (10 mg/kg po) provided 36 %
BTK occupancy and blood levels of 13 nM 5 h post dose. A rat PK of 13 revealed a
bioavailability of only 5 %, likely limited by the low permeability. Therefore we set out to
optimize the permeability, since improved oral absorption should subsequently increase target
occupancy. We envisioned that introducing a fluoro substituent ortho to the amide on the phenyl
ring occupying the H3 pocket could form an internal hydrogen bond in solution, masking the
4
6
amide NH and leading to improved permeability. The fluoro substituent was tolerated with
respect to potency and 14 indeed displayed improved permeability by more than one log unit
compared to 13. This led to a higher bioavailability of 51 % in a rat PK study and resulted in
9
6 % target occupancy after a 10 mg/kg po dose. Lowering the dose of 14 to 3 mg/kg po and 1
mg/kg po still provided BTK occupancy of 79 % and 44 %, respectively. In an effort to further
improve the potency we investigated two alternative moieties in the H3 pocket, which in
previous studies on a related reversible series (not shown) had provided a positive effect on the
potency. Surprisingly, replacing the cyclopropyl substituent in 14 with 2-hydroxypropanyl (15)
led to a decreased potency in the blood BCR/CD69 assay. Introducing a second fluoro
substituent (16) recovered the blood potency, however the permeability of 16 was poor,
ACS Paragon Plus Environment
8

Page 9 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
translating into a modest target occupancy of only 32 % after a dose of 3 mg/kg po. Based on the
promising results of 14 in the BTK occupancy assay, we profiled the compound more broadly.
However, the stability of 14 in human plasma turned out to be low (t 35 min). Plasma
1
/2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
instability could be an indication of an undesired covalent binding to plasma proteins or
enzymatic cleavage, therefore we used this assay to optimize our scaffold.
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Linker and tail modifications.
R2
HN
O
N
F
R1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
NH2
BTK
BTK IC
FcR/IL8 BCR/CD69 Permeability occupancy
5
0
R1
R2
a
b
c
d
[
nM]
IC [nM] IC [nM]
logPe pH 6.8 [%] (Dose
5
0
50
e
[
mg/kg])
O
10
11
12
3.8 ± 0.32
1.8e
11 ± 6
15 ± 4
106 ± 51
223 ± 18
n.t.
-5.0
-4.6
n.t.
N
N
n.t.
n.t.
O
O
O
O
N
3651 ± 3619 1437 ± 309
-6.2
N
1
3
3.6 ± 0.66
4.5 ± 1.6
17 ± 3
95 ± 4
<-6.7
36 (10)
O
O
9
6 (10)
O
N
14
20 ± 13
86 ± 30
-5.2
79 (3)
F
4
4 (1)
n.t.
HO
HO
O
O
N
N
1
1
5
6
23 ± 10
108 ± 78
24 ± 7
250 ± 0
55 ± 29
-6.6
O
O
F
2.3 ± 1.1
<-6.6
32 (3)
F
F
a) BTK enzyme inhibition in Caliper assay IC shown as means ± SD (n ≥ 2); b) FcγR induced
5
0
IL8 IC shown as means ± SD (n ≥ 2); c) anti-IgM/IL-4 induced CD69 expression on human
5
0
blood B cells IC shown as means ± SD (n ≥ 2); d) PAMPA permeability, logPe at pH 6.8; e)
50
BTK occupancy in rat spleen homogenate 5 h after po dose of compounds compared to vehicle
control, determined as mean (n = 3); e) n = 1; f) n.t.: not tested.
ACS Paragon Plus Environment
1
0

Page 11 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
We sought to investigate the linker to the warhead more broadly with the goal to increase the
plasma stability and in addition to further improve blood potency and in vivo PD. We combined
various linkers with the most attractive H3 pocket moiety, the fluorinated cyclopropylphenyl tail.
The propenyl linker (17) led to a two fold increase in potency in the blood BCR/CD69 assay
compared to 14 and good plasma stability (Table 2). However, the increased blood potency did
not translate into an improved PD effect, 17 performed similarly to 14 in vivo, providing 77 %
BTK occupancy, probably due to the high in vivo clearance (307 ml/min/kg). The oxygen linked
open-chain analogue 18 was in the same potency range as 14, and gave high target occupancy of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
4 % despite its only moderate permeability. Replacing the linker oxygen by a nitrogen (19) was
not tolerated leading to a roughly 10-fold loss of potency in the human blood assay. Despite a
more than two-fold reduced blood potency compared to 14, introduction of a piperidine linker
gave similar target occupancy, due to high exposure (207 nM 5 h post dose). Whereas the (R)-
pyrrolidine linker (21) was not tolerated with respect to BTK inhibition, the (S)-derivative 22
potently inhibited the blood BCR/CD69 assay and BTK occupancy of 90 % was achieved.
However, 22 suffered from a strong hERG flag (hERG dofetilide binding IC 49 ± 12 nM (n =
5
0
2
)). Replacing the pyrrolidine moiety by an azetidine provided 23, which achieved 80 % BTK
occupancy. However, as in the case of azetidine 14, plasma stability of 23 was low. An enhanced
reactivity of N-acryloyl azetidines towards cysteines has been reported and might explain the
4
7
low plasma stability of compounds 14 and 23. The corresponding (R)-azetidine 24 was less
potent than 23 in the blood assay. The N-methylated analogue of 18 (25) was nearly 5-fold more
potent than 14 in the blood BCR/CD69 assay and displayed 94 % BTK occupancy. Increasing
the length of the open chain linker to three carbons led to 26 which was significantly less potent
than 25 in the blood assay. Modifying the N-methyl substituent on the acrylamide to an ethyl
ACS Paragon Plus Environment
1
1

Journal of Medicinal Chemistry
Page 12 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
group provided 27, which was comparable to 25 with respect to blood potency and target
occupancy. However, 27 had a higher logP compared to 25 (3.6 vs 2.9) and interestingly
inhibited CYP2D6 with an IC of 1.26 M. Overall, 27 did not seem to offer any advantage
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
over 25. With the exception of azetidine acrylamide 23 all compounds from Table 2 tested for
stability in human plasma showed plasma half-lives of >120 min.
ACS Paragon Plus Environment
1
2

Page 13 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 2. Linker modifications.
O
F
HN
F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
N
N
NH2
Stability in
FcR/IL8 BCR/CD69 Permeability human
IC (nM) IC (nM) logPe pH 6.8 plasma, t [%] after a 3
BTK
occupancy
BTK IC
5
0
R1
a
b
c
d
(nM)
50
50
1/2
e
[
min]
mg/kg dose
O
1.9 ± 1.2
8.7 ± 4.6
51 ± 38
11 ± 3
44 ± 4
-4.5
-5.4
-5.8
>120
>120
n.t.
77
1
1
1
7
8
9
N
O
O
O
18 ± 13
105 ± 13
84
N
H
H
N
671 ± 690 1245 ± 350
n.t.
N
H
O
N
3
.7 ± 4.5
12 ± 2
201 ± 63
n.t.
-4.5
-3.9
>120
n.t.
77
2
2
0
1
O
O
O
N
1
290 ± 916 1869 ± 87
n.t.
O
O
N
9f
1
.4 ± 0.4
57 ± 8
-4.1
>120
90
2
2
O
N
5f
3
7
.1 ± 0.5
.5 ± 6.3
50 ± 3
-3.9
-4.1
42
80
23
O
O
N
O
60 ± 14
354 ± 59
n.t.
n.t.
2
4
ACS Paragon Plus Environment
1
3

Journal of Medicinal Chemistry
Page 14 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
1.3 ± 0.9
2.5 ± 0.7
17 ± 6.5
18 ± 5
91 ± 9
-4.3
-4.0
>120
n.t.
94
2
5
N
O
N
3
.6 ± 1.3
n.t.
26
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
5f
0.9 ± 0.2
28 ± 4
-4.2
>120
93
2
7
N
a) BTK enzyme IC shown as means ± SD (n ≥ 2); b) FcγR induced IL8 IC shown as means
± SD (n ≥ 2); c) anti-IgM/IL-4 induced CD69 expression on human blood B cells IC shown as
means ± SD (n ≥ 2); d) PAMPA permeability, logPe at pH 6.8; e) BTK occupancy in rat spleen
homogenate 5 h after po dose of compounds compared to vehicle control, determined as mean (n
5
0
50
5
0
=
3); f) n = 1; g) n.t.: not tested.
Having identified the highly potent acrylamide 25, we wanted to assess if an alternative warhead
could provide an overall advantage. Generally, a highly reactive moiety could lead to increased
binding to off-targets, whereas too low intrinsic reactivity could lower the reaction rate to the
Cys of interest. To probe the intrinsic reactivity of different warheads we investigated the
stability of the compounds towards an excess of glutathione (GSH) as a surrogate for Cys-
containing nucleophiles and proteins (Table 3). In the presence of GSH, 25 showed a slow
decrease with 68.5 % remaining parent after 24 h. For the sterically hindered methyl substituted
acrylamide 28, a significantly decreased reactivity towards GSH was observed. However, 28 was
also significantly less active in the blood BCR/CD69 assay. The 4-(dimethylamino)but-2-
enamide 29 showed a slow decrease in the presence of GSH over 24 h, similar to 25. However,
the permeability of 29 was very low (log Pe pH 6.8 <-6.5) and together with the reduced blood
BCR/CD69 potency compared to 25 we did not pursue this compound further. The propynamide
3
0 was highly unstable in the presence of GSH with complete disappearance of the parent within
2
4 h. In addition, only limited stability (t 96 min) in human plasma was observed. For the but-
1
/2
ACS Paragon Plus Environment
1
4

Page 15 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
-yneamide 31, decrease of parent was slow, but due to the decreased blood BCR/CD69 assay
potency this compound was also not considered further. In summary, compound 25 (LOU064,
remibrutinib) exhibited the best overall profile and was selected for further characterization.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
1
5

Journal of Medicinal Chemistry
Page 16 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Warhead modifications.
O
F
HN
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
N
N
NH2
Stability in
BTK IC
FcR/IL8 BCR/CD69
5
0
R1
a
b
c
presence of GSH
(nM)
IC (nM)
IC (nM)
5
0
50
d
at 24 h
O
O
1.3 ± 0.9
104 ± 77
11 ± 5
2.5 ± 0.7
18 ± 5
983 ± 617
151 ± 69
49 ± 23
68.5
95.8
60.8
0.1
25
N
O
O
263 ± 119
76 ± 3
2
2
8
9
N
O
O
N
N
O
O
1.8 ± 0.2
5.2 ± 2
6.5 ± 2
32 ± 7
30
N
O
O
161 ± 44
>20’000
79.6
3
3
1
2
N
O
O
1’300 ± 424
>10’000
n.t.
N
a) BTK enzyme IC shown as means ± SD (n ≥ 2); b) FcγR induced IL8 IC shown as means
5
0
50
±
SD (n ≥ 2); c) anti-IgM/IL-4 induced CD69 expression on human blood B cells IC shown as
50
means ± SD (n ≥ 2); d) The stability in the presence of GSH is given as ratio area (analyte/internal
standard) in %; e) n.t.: not tested.
ACS Paragon Plus Environment
1
6

Page 17 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The melting point of crystalline LOU064 is 194 °C. It has a low thermodynamic solubility of
<
0.003 g/L at pH 6.8, but solubility increases to >1.5 g/L at pH 1. The permeability is high
(PAMPA: log Pe pH 6.8 -4.3; Caco Papp(A-B) 15.9, Papp(B-A) 10.2) suggesting good
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
absorption. The half-maximum inhibition of the CYP enzymes 3A4, 2C9 and 2D6 was ≥ 5 M
(see Supporting Information). LOU064 showed some activity in an in vitro PXR reporter gene
assay (EC >5.48 M and E 94.7 %) indicating a risk for CYP induction and drug-drug
5
0
max
interactions.
LOU064 Binds Covalently to BTK. The covalent character of BTK inhibition by LOU064 was
confirmed in a series of studies. First, the kinetics of BTK inhibition were assessed in an enzyme
dilution experiment.48 The biochemical BTK enzyme assay (Supporting Information) was
modified to include a one hour preincubation of BTK protein at a concentration of 4.7 µM with a
1
0 % molar excess of compound. The compound/enzyme mixture was then diluted into kinase
assay buffer containing ATP and peptide substrate to reach a final concentration of 1 nM BTK and
.1 nM compound and enzyme activity was monitored by measuring substrate phosphorylation
Figure 4). The increase in phosphorylated substrate peptide over time indicated recovery of BTK
1
(
kinase activity by the dilution step for the DMSO solvent control, the reversible BTK inhibitor
staurosporine, as well as the inactive non-covalent ethylamide analog 32 (Table 3). In line with
expectations for a covalent binder, LOU064 showed no recovery of BTK activity after the dilution
step.
ACS Paragon Plus Environment
1
7

Journal of Medicinal Chemistry
Page 18 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Rescue of BTK enzymatic activity by dilution. The increase of phosphorylated substrate
peptide over time after the dilution step is indicated for the DMSO solvent control (black dash and
triangles), for the reversible BTK inhibitor staurosporine (blue dash and triangles), for the non-
covalent ethylamide analog 32 (red dash and empty circles) and for LOU064 (red line and filled
circles).
Secondly, we assessed covalent binding of LOU064 to the BTK kinase domain by analysis with
LC/MS (Experimental details see Supporting Information), which revealed the presence of a
main peak with 32’222.3 Da mass, closely corresponding to the predicted BTK/LOU064 1:1
adduct (based on the masses of 507.5 and 31’714.5 Da, respectively for LOU064 and the kinase
domain construct). This provided further evidence for a specific and single covalent modification
of BTK by LOU064 (Figure 5).
ACS Paragon Plus Environment
1
8

Page 19 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Deconvoluted mass spectrum of covalent BTK kinase domain (encompassing amino
acids 386-659) adduct with LOU064. The peak with a corresponding mass of 32222.3 Da is
indicative of the presence of one covalent adduct of LOU064 (507.5 Da) to the BTK kinase domain
(31’714.5 Da).
Thirdly, solving the complex structure revealed the atomic details of LOU064 binding to BTK
and confirmed the design hypothesis. As expected, the inhibitor is covalently attached to Cys481
Figure 6), with the carbonyl of the acrylamide making a hydrogen bond with the NH of the
(
backbone of Cys481. The amino-pyrimidine moiety forms a bidentate hydrogen bonding
interaction with the backbone of Met477 of the kinase hinge region. The phenyl group of LOU064
at position 4 of the 6-aminopyrimidine scaffold makes hydrophobic contacts with the Val416 side
chain while the fluorine makes a multipolar interaction with the carbonyl of Gly409 of the Gly-
49
rich loop (Figure S-1, Supporting Information). The fluoro-phenyl-cyclopropyl-group occupies
1
6
the H3 pocket and stabilizes it in an inactive state. The H3 pocket is formed by the side chains
of Gln412, Phe413 (both Gly-rich loop), Asp521, Asn526 (both part of the catalytic loop), Asp539
(DFG), Leu542, Ser543, Val546, and Tyr551 (belonging to the activation loop) (Figure S-1). The
ACS Paragon Plus Environment
1
9

Journal of Medicinal Chemistry
Page 20 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fluorine in this pocket points towards the cavity, instead of towards the NH of the amide of the
ligand, most likely to avoid the unfavorable vicinity to the carbonyl of Asn526 (Figure S-1) .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. X-ray complex structure of BTK kinase domain bound to LOU064 (PDB 6TFP, protein
chain A with ligand chain L). The molecular surface of LOU064 is colored in orange and key
residues lining up the binding site are shown as labelled stick model. The main polar interactions
between protein and inhibitor are indicated with dotted green lines. For improved clarity water
molecules are omitted.
Selectivity of LOU064. Our aim was to combine covalent BTK binding and the high selectivity
associated with binding to the inactive BTK conformation. The selectivity of LOU064 was
assessed not only in a selected panel of enzymatic kinase assays, but also in a competition binding
ACS Paragon Plus Environment
2
0

Page 21 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
50
assay against 395 human non-mutant kinases and 61 atypical, mutant or pathogen kinases. In this
screen (KINOMEscan™, DiscoverX), LOU064 at a concentration of 1 M showed full inhibition
of probe binding to BTK (0.25 % remaining probe binding) and partial inhibition of TEC (24 %
remaining probe binding) and BMX (53 % remaining probe binding), indicating an excellent
kinase selectivity profile (TREEspotTM Kinase Dendogram of LOU064 (Figure S-2) and
KINOMEscan data in Supporting Information).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Based on this kinome-wide screen, the selectivity of LOU064 was further assessed and compared
to clinical covalent BTK inhibitors. As the determination of IC values in biochemical enzyme
5
0
4
8
assays for covalent inhibitors suffers from time-dependent effects we determined the binding
50,51
constants (K ) in a competition binding assay
for a set of Cys-containing kinases (Table 4).
d
LOU064 showed very potent affinity to BTK with a subnanomolar K of 0.63 nM and with a
d
selectivity of 175 fold against TEC (K of 110 nM) and 857 fold against BMX (K of 540 nM).
d
d
LOU064 did not show binding to ITK, EGFR, ERBB2, ERBB4 and JAK3 up to 10 M. By
contrast, ibrutinib (1) showed similar high affinity for BTK, but poor selectivity against BMX,
TEC, EGFR, ERBB2 and ERBB4. Of note, acalabrutinib (2), tirabrutinib (3) and evobrutinib (4)
are significantly less potent than LOU064 on BTK and less selective. Branebrutinib (5)
displayed high affinity for BTK, comparable to LOU064 and ibrutinib, with an improved
selectivity profile compared to ibrutinib, however inferior to LOU064, potently inhibiting BMX
3
9
and TEC. These binding data are in line with recent publications in other assay systems and our
data in biochemical enzyme assays (not shown). Overall, in addition to being amongst the most
potent inhibitors for BTK, LOU064 differentiates with its excellent, best-in-class kinase
selectivity from other clinical-stage covalent inhibitors.
ACS Paragon Plus Environment
2
1

Journal of Medicinal Chemistry
Page 22 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Binding constants (K in nM) of covalent BTK inhibitors for selected kinases containing
a conserved cysteine and selectivity vs off-target kinases (Kd, off-target/Kd, BTK).
d
BTK BMX TEC
ITK
EGFR ERBB2 ERBB4 JAK3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Kd
Fold
Kd
0.63
1
540
857
1.6
0.6
610
29
110 >10000 >10000 >10000 >10000 >10000
175 >15873 >15873 >15873 >15873 >15873
2
5
(
LOU064)
1.9
1
1.7
0.9
14
57
30
6.9
3.6
1.2
0.6
2.4
1.3
140
6.7
37
19
1
(ibrutinib)
Fold
Kd
21
1
>10000
>476
5600
267
7.4
>10000
>476
2
(
acalabrutinib)
Fold
Kd
0.7
0.4
14
1
47
6.2 >10000 >10000
610
>10000 >10000
3
(
tirabrutinib)
Fold
Kd
3.4
31
0.4
4.5
>714
3700
231
>714
7100
443
44
>714
5600
350
>714
>10000
>625
16
1
>10000
>625
4
(
evobrutinib)
Fold
Kd
1.9
22
0.3
0.56
1
0.99
1.8
320
>10000 >10000
>17857 >17853
2500
4464
1300
5
(branebrutinib)
Fold
39
571
2321
We evaluated the cellular selectivity of LOU064 in a panel of culture systems with primary
human cells representative for several tissues, including fibroblasts, epithelial cells, keratinocytes
5
2
and immune cells (BioMAP Diversity plus panel, DiscoverX). LOU064 was tested at
concentrations of 270, 900, 3000 and 10000 nM, all concentrations well above its cellular BTK
inhibition in cell culture conditions (Figure S-3, Supporting Information). The main activity
ACS Paragon Plus Environment
2
2

Page 23 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
detected was in the BT panel, and represents the intended inhibition of cellular BTK function in
B cells stimulated via the B cell receptor and in the presence of T cell co-stimulatory pathways,
resulting in the inhibition of several inflammatory readouts such as TNF-α, IL-17A, IL-6 and IL-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
, as well as inhibition of B cell proliferation (indicated by the gray arrow in the BT panel). The
response was not concentration dependent as all concentrations tested were well above maximal
cellular BTK inhibition. Of note is the absence of a peak in the EGFR signal in the HDF3CGF
panel, clearly indicating the lack of any activity of LOU064 in the dermal fibroblast system, an
effect which has been shown for BTK inhibitors with off-target effects on EGFR, such as
2
2
ibrutinib (Figure S-4, Supporting Information). Across all these cellular systems, LOU064
showed no cytotoxicity. In addition to the expected effects on B cell proliferation, LOU064
exerted effects on fibroblast proliferation at concentrations of 270 and 10000 nM (gray arrow in
the HDF3CGF panel).
Furthermore, the high selectivity of LOU064 was confirmed by testing against a broad panel of
in vitro assays (in binding or in functional mode) comprising 85 G-protein coupled receptors,
transporters, nuclear receptors, enzymes and ion channels (Supporting Information). LOU064
did not show binding, inhibition, or activation in any of these assays at concentrations up to 10
µM, with the exception of PIK4CB (IC 3.1 µM) and Bile Salt Export Pump (BSEP, IC 6.6
5
0
50
µM), thus establishing a more than 2000-fold selectivity for BTK over these off-targets. In the in
vitro hERG patch clamp assay an IC of 1.4 M was measured. In preclinical invasive and non-
50
invasive dog telemetry studies after single and long-term dosing, no effect on any cardiovascular
parameter was detected. As the non-covalent analog 32 showed very similar activity on BSEP
(
IC 3.7 µM) and hERG (IC 2.9 µM in Qpatch assay), it is likely that the interaction of
50 50
LOU064 with these off-target proteins is non-covalent. No significant inhibition of other cardiac
ACS Paragon Plus Environment
2
3

Journal of Medicinal Chemistry
Page 24 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ion channels (Nav1.5, Cav1.2, KCNQ1) was observed and LOU064 was found negative in
genotoxicity and mutagenicity assays (in vitro Ames, in vitro and in vivo micronucleus tests).
Similarly, LOU064 was negative in the 3T3 NRU phototoxicity assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Preclinical PK Profile of LOU064. The in vitro clearance of LOU064 in microsomes was
moderate in rat, and high in mouse and dog (Table 5). The in vivo pharmacokinetic properties of
LOU064 were assessed in rat, mouse and dog (Table 5). In rats, the compound displayed a
moderate clearance, a low volume of distribution, a short half-life, and a moderate bioavailability.
LOU064 was mainly cleared via oxidative metabolism and elimination of unchanged LOU064
was not a major pathway in rat. In mice, LOU064 displayed a similar PK profile as for rats with
high CL, a low volume of distribution and a short half-life. In dogs, the clearance was high, in the
range of the hepatic blood flow, the volume of distribution was low, the half-life was short and the
bioavailability was low. Formulations were developed which enabled preclinical toxicology
studies to be carried out (not shown).
Table 5. In vitro and in vivo pharmacokinetic parameters of LOU064 in rat, mouse and dog
Rat
33
Mouse
325
Dog
146
In vitro CL int (L/min/mg)
Hepatic extraction (%)
52
93
87
Female
Lewis
Female
Male
In vivo
C57BL/6
Beagle
Dose
1 mg/kg iv
1 mg/kg iv
1 mg/kg iv
ACS Paragon Plus Environment
2
4

Page 25 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
mg/kg po
44 ± 12
3 mg/kg po
97 ± 11
3 mg/kg po
29 ± 4
1.0 ± 0.1
0.9 ± 0.1
19
CL (mL / min / kg)
V (L / kg)
ss
1.4 ± 0.3
1.0 ± 0.1
1.0 ± 0.1
53 ± 14
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
t (h)
0.5 ± 0.03
148 ± 33
0.3
1
/2
Cmax d.n. (nM)
Tmax (h)
0.3 ± 0.1
16 ± 4
0.8
F (%)
29 ± 3
5
AUC po d.n. (nM · h)
225 ± 27
54 ± 15
55
In essence, the preclinical ADME properties of LOU064 are characterized by excellent
permeability, high clearance and fast elimination.
In vitro BTK Inhibition Kinetics. To further characterize the mechanism of BTK inhibition by
LOU064, we tested the kinetics of enzymatic inhibition. In biochemical assays of enzymatic
activity with recombinant full-length human BTK, LOU064 showed very fast inhibition of BTK
(
Figure S-5, Supporting Information). LOU064 showed potent BTK inhibition starting from the
earliest time-point and no indication of a time-dependent shift in IC over the timeframe that
5
0
allowed reliable detection of BTK activity or inhibition. The very fast binding kinetics of LOU064
to purified BTK prevented the determination of accurate and meaningful biochemical reaction
parameters.
We therefore assessed the engagement of cellular BTK in a clinically relevant matrix - human
blood. Blood from healthy volunteers was mixed with serial dilutions of LOU064, incubated at
ACS Paragon Plus Environment
2
5

Journal of Medicinal Chemistry
Page 26 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
room temperature to allow binding to cellular BTK and at given time points, aliquots of blood
were sampled and analyzed for covalent binding of LOU064 to BTK with immunoassays for free
BTK (i.e. not covalently occupied by compound) and total BTK protein (see Supporting
Information). LOU064 showed very rapid covalent binding to cellular BTK in human blood at
concentrations as low as 300 nM (Figure 7, left panel).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Time- and concentration-dependent in vitro engagement of cellular BTK by LOU064
and related inhibitors in human blood. Left panel shows data for LOU064 from two independent
experiments with different blood donors (solid lines show non-linear one-phase association curve
fits). Right panel shows the association kinetics for a concentration of 300 nM for each compound
(solid lines show non-linear one-phase association curve fits). Individual data from two
independent experiments with different blood donors.
We compared the binding characteristics of LOU064 to related covalent BTK inhibitors in the
same type of assay at a concentration of 300 nM (Figure 7, right panel). In contrast to the rapid
and complete BTK binding of LOU064, the other covalent BTK inhibitors, with exception of
branebrutinib, showed slower binding kinetics. A comparison of the enzymatic and cellular
ACS Paragon Plus Environment
2
6

Page 27 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
potency of LOU064 to these covalent BTK inhibitors across several assays confirmed the very
high potency of LOU064 (Table 6). These data suggest that LOU064 has very fast reaction kinetics
and rapidly reaches full BTK inhibition in relevant matrices such as human blood.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Comparative potency of LOU064 and covalent BTK inhibitors
BTK
FcR/IL8
BCR/CD69
FcεR/CD63
IC50 (nM)d
a
b
c
IC (nM)
IC (nM)
IC (nM)
5
0
50
50
LOU064 (25)
Ibrutinib (1)
1.3 ± 0.9
2.3 ± 2
19 ± 22
5.2 ± 5
62 ± 4
2.5 ± 0.7
16 ± 14
18 ± 5
66 ± 57
203 ± 77
132 ± 46
320 ± 74
18 ± 4
67 ± 54
230 ± 210
747 ± 75
363 ± 200
1182 ± 819
40 ± 10
Acalabrutinib (2)
Tirabrutinib (3)
Evobrutinib (4)
Branebrutinib (5)
24 ± 24
26 ± 16
61 ± 37
0.1 ± 0.0518
6.0 (n = 1)
a) BTK enzyme inhibition in Caliper assay IC shown as means ± SD (n ≥ 2); b) FcγR induced
50
IL8 IC shown as means ± SD (n ≥ 2); c) anti-IgM/IL-4 induced CD69 expression on human
5
0
blood B cells IC shown as means ± SD (n ≥ 2); d) anti-IgE-induced degranulation and CD63
5
0
expression on human blood basophils.
In vivo BTK Engagement. For a covalent inhibitor of BTK, the PD effects are determined by
the extent and duration of BTK target occupancy. The initial inactivation of BTK by LOU064
binding is primarily determined by the systemic exposure of the drug. Once the drug is eliminated
from systemic circulation the duration of BTK inhibition becomes independent from drug
exposure and is determined by the turnover of the inactive drug/protein complex by synthesis of
5
3
new BTK protein. For BTK, the turnover of inactivated protein by newly synthesized protein has
been described in preclinical models and ranges from 20 – 40 % of BTK protein being
resynthesized after 24 h.18,22,54
ACS Paragon Plus Environment
2
7

Journal of Medicinal Chemistry
Page 28 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We set out to determine the detailed PK/PD relationship of LOU064 in rats by using BTK
occupancy as the primary PD readout. A series of studies were performed in rats to determine the
dose range leading to varying degrees of BTK occupancy in spleen (Figure 8). To minimize the
impact of circulating LOU064, the animals were killed 5 hours after oral dosing and tissues were
sampled. BTK occupancy in spleen homogenates was measured relative to vehicle controls. A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dose-response curve fit indicates an EC of 0.6 mg/kg (EC 1.6 mg/kg). The analysis of LOU064
50
90
in blood from these animals showed that for the 1 mg/kg dose, resulting in an average of 78 %
BTK occupancy, the systemic exposure was 49.1 nM at 0.5 hours post dose (5.6 nM at 5 hours).
This suggests that very low and transient systemic exposures of LOU064 are sufficient for BTK
inhibition.
Figure 8. Spleen BTK occupancy relative to vehicle controls was measured 5 hours after oral
dosing in female rats combined from two separate studies (with 3 animals per each dose group).
Open triangles show the BTK occupancy levels in the combined vehicle dosed animals. Filled
ACS Paragon Plus Environment
2
8

Page 29 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
circles show the BTK occupancy of LOU064 treated animals. The dotted line shows a dose-
response curve fit.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Preclinical Efficacy in SRBC and CIA Models. To extend the preclinical PK/PD relationship
to repeated dosing regimen, we examined the efficacy of LOU064 in a model of B cell dependent
55
antibody formation following immunization with sheep red blood cells (SRBC). Female rats
were immunized with SRBC and treated for 4 days with one daily oral dose of LOU064 starting
on the day of immunization. On day 5, SRBC antibody formation was assessed ex vivo on
splenocytes, as well as BTK occupancy in the same tissue (Figure 9, left panel). The IgM antibody
response was maximally inhibited (by 96 %) at a dose of 3 mg/kg q.d.. This level of IgM response
inhibition was correlated to a spleen BTK occupancy at the 24 h trough of 71.5 % (Figure 9, right
panel). Doses as low as 0.3 mg/kg demonstrated a partial, but significant inhibitory effect on SRBC
specific antibody response (with a trough BTK occupancy in spleen of 31 %).
ACS Paragon Plus Environment
2
9