Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases

Article abstract of DOI:10.1021/acs.jmedchem.9b00794

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Full text of DOI:10.1021/acs.jmedchem.9b00794

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Drug Annotation
Discovery of Evobrutinib: An Oral, Potent and Highly
Selective, Covalent Bruton’s Tyrosine Kinase (BTK)
Inhibitor for the Treatment of Immunological Diseases
Richard Caldwell, Hui Qiu, Ben C Askew, Andrew T Bender, Nadia Brugger, Montserrat Camps,
Mohanraj Dhanabal, Vikram Dutt, Thomas Eichhorn, Anna S Gardberg, Andreas Goutopoulos,
Roland Grenningloh, Jared Head, Brian Healey, Brian L Hodous, Bayard R Huck, Theresa L Johnson,
Christopher Jones, Reinaldo C Jones, Igor Mochalkin, Federica Morandi, Ngan Nguyen, Michael
Meyring, Justin R Potnick, Dusica Cvetinovic Santos, Ralf Schmidt, Brian Sherer, Adam Shutes, Klaus
Urbahns, Ariele Viacava Follis, Ansgar A Wegener, Simone C. Zimmerli, and Lesley Liu-Bujalski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00794 • Publication Date (Web): 01 Aug 2019
Downloaded from pubs.acs.org on August 1, 2019
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Discovery of Evobrutinib: An Oral, Potent and
Highly Selective, Covalent Bruton’s Tyrosine
Kinase (BTK) Inhibitor for the Treatment of
Immunological Diseases
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†
Richard D. Caldwell,* Hui Qiu,* Ben C. Askew, Andrew T. Bender, Nadia Brugger,
‡
Montserrat Camps, Mohanraj Dhanabal, Vikram Dutt, Thomas Eichhorn, Anna S.
§
¶
Gardberg, Andreas Goutopoulos, Roland Grenningloh, Jared Head, Brian Healey,
‖
Brian L. Hodous, Bayard R. Huck, Theresa L. Johnson, Christopher Jones, Reinaldo C.
††
‡‡
§§
¶¶
Jones, Igor Mochalkin, Federica Morandi, Ngan Nguyen, Michael Meyring, Justin
R. Potnick, Dusica Cvetinovic Santos, Ralf Schmidt, Brian Sherer, Adam Shutes,‖‖
Klaus Urbahns, Ariele Viacava Follis, Ansgar A. Wegener, Simone C. Zimmerli, and
Lesley Liu-Bujalski
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ADDRESS: EMD Serono Research & Development Institute, Inc., 45 A Middlesex
Turnpike, Billerica, MA 01821, USA (a business of Merck KGaA, Darmstadt, Germany).
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KEYWORDS: BTK, covalent, selective, evobrutinib, kinase
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ABSTRACT: Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent
role in the treatment of B cell malignancies. However, further refinement is needed to
this class of agents, particularly in terms of adverse events (potentially driven by kinase
promiscuity) which preclude their evaluation in non-oncology indications. Here, we
report the discovery and preclinical characterization of evobrutinib, a potent, obligate
covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical
pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo
evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over
epidermal growth factor receptor and other Tec family kinases suggested a low potential
for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in
several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and
systemic lupus erythematosus.
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INTRODUCTION
Bruton’s tyrosine kinase (BTK) is a member of the Tec family of tyrosine kinases, and is
1
expressed in B cells, macrophages, and monocytes, but not in T cells. BTK plays a
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crucial role in signaling through the B cell receptor (BCR) and the Fcγ receptor (FcγR) in
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B cells and myeloid cells, respectively. Indeed, mutations in the human Btk gene can
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result in the B cell specific immunodeficiency, X-linked agammaglobulinemia, which is
characterized by a block in pre-B cell differentiation; this leads to a lack of B cells and
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plasma cells, and markedly reduced levels of serum immunoglobulins. Likewise, Btk
mutations in mice with X-linked immunodeficiency, and targeted deletion of Btk in
knockout mice result in defects in B cell development and proliferation.3,4 However,
pharmacological inhibition of BTK has not resulted in depletion of B cells in preclinical
5
models. Consequently, BTK is considered a promising therapeutic target for the
treatment of various diseases involving B cell and/or macrophage activation, such as B
cell malignancies, asthma, rheumatoid arthritis, systemic lupus erythematosus, and
multiple sclerosis.6,7
The small molecule irreversible BTK inhibitor, ibrutinib, which covalently targets
Cys481 within the ATP-binding pocket, is approved for the treatment of mantle cell
lymphoma (MCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma,
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Waldenström’s macroglobulinemia, and marginal zone lymphoma, as well as graft
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versus host disease. Ibrutinib demonstrated clinical efficacy in previously difficult-to-
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treat lymphomas such as treatment-resistant/refractory CLL.8,9 However, this first in
class BTK inhibitor has also shown side effects, including bleeding, rash, diarrhea, and
atrial fibrillation, which have been attributed, in part, to off-target kinase-inhibitory
related effects on the epidermal growth factor receptor (EGFR) and other Tec family
kinases.10-13
With the aim of improving the overall tolerability of BTK inhibitors while
maintaining the efficacy of ibrutinib, new BTK inhibitors are being developed that have
13
more refined pharmacologic profiles. Additionally, these agents are designed to
circumvent the inhibitory effect of ibrutinib on antibody-dependent cell-mediated
cytotoxicity, which may be important for the activity of other oncology therapies
13
administered concomitantly with a BTK inhibitor. In 2017, acalabrutinib became the
initial second-generation, covalent BTK inhibitor to be approved by the FDA, for the
treatment of MCL.9,14 However, for chronic autoimmune indications, clinical
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development progress has lagged behind that of oncology indications. Recently, there
has been increased interest in the development of BTK inhibitors in the
autoimmune/inflammatory arena.
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Herein, we describe work which led to the identification of the potent and
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irreversibly covalent BTK inhibitor, evobrutinib (A18, M2951) for the treatment of
autoimmune diseases. Evobrutinib has excellent kinome selectivity, particularly against
EGFR, an acceptable preclinical pharmacokinetic/pharmacodynamic (PK/PD) profile,
and is efficacious in in vivo efficacy models of rheumatoid arthritis. Evobrutinib is being
evaluated in clinical development for treatment of various immunological diseases.16
RESULTS AND DISCUSSION
Medicinal Chemistry Strategy
The target profile for a development candidate for chronic administration in autoimmune
diseases included potency, high BTK kinase-selectivity, particularly against EGFR, and
moderate clearance. It was hypothesized this profile would result in a low daily dose
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which should minimize the risk of liver damage; as covalent binding, combined with a
high daily dose, has been reported to be linked to drug-induced liver injury.17
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To achieve BTK potency and kinase selectivity simultaneously, we employed
multiple strategies. First it was desired to have an obligate type covalent inhibitor, in
which the covalent binding is essential for the potency of the molecule. Additionally,
targeting Cys481 with a warhead to form a covalent bond enhances selectivity because
only 10 other kinases: BLK, BMX, EGFR, ERBB2, ERBB4, ITK, JAK3, TEC, MKK7, and
TXK contain cysteine in the same region of the receptor. Finally, interaction with the
gatekeeper Thr474 further increases selectivity as only 20% of all human kinases have
threonine as a gatekeeper residue.18,19
Identification of a Novel Hinge Binder
Work to identify suitable chemical starting points began with analysis of the X-ray crystal
structure of B43 (Figure 1, PDB code: 3gen).18,20 B43 is a moderately potent inhibitor of
BTK and occupies the ATP binding pocket of the BTK kinase domain. The amino-
pyrimidine group, acting as a hinge binder, forms interactions with the gatekeeper
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residue Thr474, as well as backbone carbonyl and amide N interactions with Glu475
and Met477, respectively. The O-linked phenoxy phenyl group occupies the long and
narrow selectivity pocket adjacent to gatekeeper Thr474, and the lipophilic cyclopentyl
group sits in the ribose pocket, which is near Cys481, providing an indication of the
proper vectors and angles for the desired covalent connection.
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Figure 1. X-ray Structure of BTK Ligand B43 Bound to the BTK Kinase Domain.
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Structure–activity relationship (SAR) exploration focused on replacing the
pyrrolopyrimidine of B43 with other similarly substituted hinge binding moieties bearing aryl
linkers appended with an acrylamide warhead. This work revealed that several hinge binding
moieties were tolerated for potent BTK inhibitory activity (Table 1). The pyrimidine analog A3
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showed excellent inhibition of BTK in a biochemical assay, while the removal of the 2-amino
moiety led to nearly 10-fold loss in potency in compound A4. Pyridine analog A5 also showed
excellent BTK inhibitory activity, and both A3 and A5 showed 25- to 30-fold more potent
inhibition of BTK versus EGFR. However, A5 had unacceptable activity in the hERG
(
human ether-a-go-go-related gene) channel assay (Supporting Information). In contrast
to A3 and A5, pyrimidine analogs such as A7, with opposite orientation of the hinge
binder, did not provide an acceptable ratio of inhibition of BTK over EGFR and were
subsequently deprioritized. In addition, analysis of the crystal structures of A5 and A7
demonstrated that these hinge binders both achieve interactions in the identified hinge
region while maintaining the critical vectors and angles required to effectively engage
the back pocket and Cys481 as covalent BTK inhibitors (Figure 2). Finally, attaching the
linker to the hinge binder through an N atom as in compound A8 also provided good
inhibition of BTK but was equipotent at inhibiting EGFR. Based on these data, the
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amino-pyrimidine hinge binder with the amino moiety oriented adjacent to gatekeeper
residue Thr474, as in A3 and A5, was selected for further exploration.
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Table 1. SAR for Hinge-Binder Optimization
O
NH
O
A =
B =
BTK enzymatic
IC50 (nM)
hERG
Compound
Hinge-binder group
EGFR IC50 (nM)
% inhibition at 10 µM
B43
N
3
10
177
--
A
H N
N
N
2
B
N
A3
A4
O
A
3
78
--
3%
H N
N
2
B
O
A
2
3
46%
N
N
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B
A5
A6
A7
O
N
A
5
140
81%
H N
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B
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O
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--
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--
N
A
O
B
8
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--
N
N
NH2
B
A8
HN
N
A
4
--
N
H N
2
hERG, human ether-a-go-go-related gene; IC50, half maximal inhibitory concentration; Ki, inhibitory constant; SAR,
structure–activity relationship
Figure 2. Overlay of Crystal Structures of A5 and A7
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2
3
4
5
6
7
8
9
0
Optimization of the Linker
Further characterization of the hit molecule A3 showed that, although it demonstrated
2
1
good potency in the human peripheral blood mononuclear cell (PBMC) assay ( IC =
50
9.9 nM), this molecule had poor solubility (< 2.4 µM) (Table 2), and poor metabolic
stability (high intrinsic clearance, Cl =277 µL/min/mg) in human liver microsomes.
int
Subsequent efforts focused on modification of A3 to improve solubility and metabolic
stability. Preliminary SAR and X-ray co-crystal information suggested that the
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
4
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4
4
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4
5
5
5
5
5
5
5
5
5
5
6
modification of the linker moiety might be amenable for addressing low solubility of the
series. Replacing the flat aromatic ring in compound A3 with a saturated moiety, such
as the di-amino cyclohexane ring in analog A9, showed good improvement in solubility
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
versus A3 (134 µM vs 2.4 µM) and metabolic stability (Cl =27 vs 277 µL/min/mg),
int
without significant loss in enzyme potency.
Various saturated linkers were evaluated with the goal of improving potency in
the enzymatic assay (<10 nM). Modifications such as moving the amino group around
the cyclohexyl ring, changing the size of the ring, or adding a bridge unit into the linker,
maintained the high solubility, however, did not lead to any meaningful boost in potency
for A10–A12. Continued exploration of the linker moiety showed that attachment of the
linker to the hinge binding core via a secondary amine, such as in compound A13, was
not tolerated. It was hypothesized that this substitution caused steric collision between
the linker and the protons at the proximal phenyl ring which altered the vector of the
linker towards the Cys481. In contrast, analog A14, with the reversed linker, was 220-
fold more potent in enzymatic assay, but suffered from a poor human microsomal
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stability. Additional exploration revealed that the pyrrolidine linker in A15 was less well
tolerated. Introduction of a spiral linker boosted the potency in molecule A16.
Unfortunately, A16 showed poor microsomal stability and an unacceptable hERG
profile.
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
Shifting the N atom in A14 as depicted in A17 led to a more than 200-fold
reduction in enzymatic potency (IC 1510 nM for A17 vs 6.5 nM for A14). Insertion of
50
one methylene group into A17 led to the discovery of A18 (evobrutinib). Evobrutinib A18
showed excellent potency in the biochemical and PBMC assays, favorable permeability
in the Caco-2 assay, good solubility and microsomal stability, and acceptable hERG
inhibition (see Supporting Information for assay methods). Finally, a hydroxyl group was
added in molecule A19 to reduce lipophilicity.
Table 2. SAR for Linker Optimization
O
O
X
N
H N
2
N
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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
Compound
X
BTK
Caco-2
Caco-2
Kinetic
solubility
(µM)
hERG K
i
HLM Clint
(µL/min/mg
prot.)
(
linker)
enzymatic/PBMC
IC50 (nM)
P
app (A-B)
Efflux ratio
(nM) or %
group
(×10-6 cm/s)
inhibition at
1
0 µM
H
N
A3
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
.9/9.9
1.6
4.2
4.3
1.6
3.4
<2.4
16
3%
277
43
O
H
N
A8
A9
4
.0/19
530
NH
H
N
1
4/--
11.9
134
1800
27
cis
NH
NH
A10
A11
A12
33/660
10
3.7
1.5
1.4
154
158
110
--
--
41
202
36
cis
NH
HN
2
1/--
23.6
21.7
cis
NH
HN
NH
9
.9/70
1300
H
N
A13
1
500/--
--
--
--
--
--
N
N
A14
A15
6
5
.5/143
.5/217
--
--
--
43%
40%
598
77
NH
N
25.2
1.0
160
NH
N
A16
2.1/66
9.3
2.1
153
780
777
NH
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9
N
A17
1
510/--
--
22
9
--
--
--
--
NH
N
A18
8
.9/61
.9/26
1.2
3.7
60
3100
45%
110
68
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
NH
N
A19
5
169
HO
NH
Clint, intrinsic clearance; HLM, human liver microsomes; PBMC, peripheral blood mononuclear cell
Back Pocket Optimization
Additional modifications directed to the back pocket to further improve microsomal
stability of A18 (evobrutinib) proved to be unsuccessful (Table 3). Substitutions at the
proximal phenyl ring (A20, A21) resulted in a dramatic loss in potency on BTK inhibitory
activity. No improvement in metabolic stability was observed from modifications of the
distal phenyl ring (A22-A25). Only analogs A23 and A24 displayed similar BTK inhibition
compared to A18, both with no obvious improvement in clearance. Subsequently, efforts
to optimize the back pocket were not pursued further.
Table 3. SAR for Selectivity-Pocket Optimization (Proximal and Distal Ring Modifications)
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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
HN
N
R1
N
O
N
H N
2
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
Compound
R
1
group (selectivity pocket)
BTK enzymatic
IC50 (nM)
HLM Clint
(µL/min/mg)
A18
O
O
8
.9
110
evobrutinib
CF3
A20
A21
A22
>
100,000
--
O
F
F
1
60
143
143
O
150
18
F
A23
A24
A25
O
O
145
199
688
F
9
.0
O
730
N
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Page 19 of 53
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O
A26
N
H
1200
--
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
BTK, Bruton’s tyrosine kinase; Clint, intrinsic clearance; HLM, human liver microsomes; IC50, half maximal
inhibitory concentration.
Warhead Optimization
Identification of an optimal warhead to engage the Cys481 residue was driven by the
goal of designing an obligate covalent inhibitor of BTK. A diverse array of warheads was
evaluated to optimize potency, reactivity and plasma stability, and to identify a suitable
covalent inhibitor for further in vivo studies (Table 4). Analogs bearing an acrylamide
1
9
warhead such as that of ibrutinib displayed acceptable biochemical and cellular
potency, as exemplified in analog A18. Replacement of the acrylamide of A18 by the
acetamide moiety in compound A27 resulted in an analog with no significant BTK
inhibitory activity, demonstrating the requirement of a warhead for efficient inhibition of
BTK. Compounds such as A28 – A30, bearing warheads that are theoretically highly
reactive22,23 did show excellent enzyme inhibitory activity, but suffered from
unacceptable plasma stability. These results were attributed to the high reactivity of the
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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
warheads. It was speculated that these compounds were binding non-specifically to
cellular components before they were able to reach the target. Substitution with Michael
acceptors that are theoretically less reactive than acrylamide22,23 led to a decrease in
BTK inhibitory activity compared to compound A18, as seen with analog A32. Based on
these data, compound A18, bearing an acrylamide warhead, possessed the optimal
combination of potency, reactivity, and plasma stability to be selected for additional
profiling.
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 obligatory nature of covalent inhibition with A18 was further confirmed by
comparing its activity with that of A27 against a mutant BTK kinase domain with a
cysteine to serine substitution, which resulted in a lack of covalent binding by either
molecule. Both compounds inhibited the BTK mutant with similarly low potency (IC50
8.6–8.7 µM), reflecting their reversible affinities for the BTK kinase domain without any
covalent binding. Thus, while A27 showed minimal inhibitory activity regardless of
Cys481 binding, the approximately 1,000-fold increase in potency of A18 against the
wildtype BTK kinase domain demonstrated that its potency is derived from covalent
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engagement of Cys481. Both A18 and A19 were further characterized in PK/PD studies
in mice.
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 4. SAR for Warhead Optimization
BTK
enzymatic/PBMC
K
irrev = Kinac/Ki
% remaining in
plasma at 4 h
Compound
R
1
group
IC50 (nM)
(1/Ms)
A18
8
.9/61
63,000
81
--
evobrutinib
A27
>
10000/--
--
A28
0
.8/45
113,000
16
A29
0
.6/160
--
49
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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
A30
A31
0
.2/--
--
--
34
--
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
90
PK/PD Studies
To confirm whether the in vitro effects with A18 and A19 translated into B cell inhibition
in vivo, these compounds were tested head-to-head in a B cell stimulation assay in
24
normal C57BL/6 mice. Both compounds were dosed at 1 mg/kg orally.
Compound A19 inhibited B cell activation by 42% and 15% at 1 and 24 h after
dosing, respectively (Figure 3). Compound A18 (evobrutinib) achieved a greater B cell
inhibition of 71% and 25% at 1 and 24 h. This was despite a lower maximum plasma
concentration (Cmax) of A18 compared to A19 (52 nM versus 257 nM). These data
showed that A18 was a more potent B cell inhibitor than A19 in vivo.
Figure 3. PK/PD Studies in Mice
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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
Synthesis of Evobrutinib (A18)
The synthesis of evobrutinib (A18) began with regioselective nucleophilic substitution of
the 6-chloride of 5,6-dichloropyrimidin-4-amine by the primary amine of tert-butyl
4-(aminomethyl)piperidine-1-carboxylate, which led to generation of tert-butyl 4-{[(6-
amino-5-chloropyrimidin-4-yl)amino]methyl}piperidine-1-carboxylate (Figure 4). Subsequent
Suzuki coupling of this chloropyrimidine intermediate with phenoxyphenylboronic acid
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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
yielded the molecule tert-butyl 4-({[6-amino-5-(4-phenoxyphenyl)pyrimidin-4-
yl]amino}methyl)piperidine-1-carboxylate. Deprotection under acidic conditions and
subsequent neutralization afforded the piperidine derivative 5-(4-Phenoxy-phenyl)-N-
piperidin-4-ylmethyl-pyrimidine-4,6-diamine. Finally, the acrylamide was introduced to
give the target compound N-[(1-acryloylpiperidin-4-yl)methyl]-5-(4-
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
phenoxyphenyl)pyrimidine-4,6-diamine (A18; evobrutinib; step d).
Figure 4. Synthetic Route for Evobrutiniba
O
O
O
O
O
O
H
N
N
N
N
N
N
Cl
O
O
Cl
NH
NH
NH2
a
NH
N
Cl
b
c
N
N
NH2
N
NH2
N
NH2
N
NH2
1
2
3
4
O
N
N
O
d
NH
N
NH2
A18
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a(a) DBU, DMF, 90 °C, 91%; (b) 4-phenoxylphenyl boronic acid, Pd(OAc)
, S-phos, K
CO
, dioxane-H O
2
2
3
2
(10:1 v/v), 150 °C, 48%; (c) HCl, MeOH, rt; (d) acryloyl chloride, NaHCO
3
, H O-THF (1:10, v/v), rt, 9%
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
Characterization of Evobrutinib: Biophysical Confirmation of Covalent, Irreversible
Binding
Covalent binding to Cys481 is confirmed by analysis of the X-ray crystal structure of
evobrutinib bound to the BTK kinase domain, showing continuous electron density between
Cys481 and the evobrutinib warhead (Figure 5).
Figure 5. Crystal Structure of Evobrutinib Bound to the BTK Kinase Domain
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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
Mass Spectrometry
Reversed-phase (RP)–LC–MS analysis of intact BTK and BTK plus evobrutinib further
confirmed the covalent nature of evobrutinib binding and indicated 1:1 stoichiometry (Figure S1
in the Supporting information). Thus, evobrutinib is bound to only one cysteine residue on the
BTK kinase domain. Orthogonal confirmation of evobrutinib–BTK covalent binding with
1:1 stoichiometry was also achieved by isothermal calorimetry (see Figure S2 in the
Supporting Information).
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Jump Dilution Assay
BTK IC was measured routinely for each compound to drive medicinal chemistry
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optimization. In addition, a jump dilution assay was used to qualitatively check the
irreversible nature of key compounds (see Supporting Information). Lack of recovery of
enzymatic activity was consistent with irreversible inhibition of BTK by evobrutinib
(Figure S3). In general, time-dependent BTK IC correlated well with the K /K ratio
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inact
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(
where K is the affinity of initial non-covalent binding, and K
is the maximum
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potential rate of enzyme inactivation), which represents the second-order rate constant
for the reaction of evobrutinib with BTK (Figure S4 and Table S1 in the Supporting
Information).25
Evobrutinib Kinase Selectivity
The high kinase selectivity of evobrutinib is derived from multiple factors. Its obligate
covalent binding to a rare cysteine residue and interaction with a unique threonine
gatekeeper-mediated selectivity pocket in structurally related kinases accounts for much
of the agent’s high kinase selectivity. For kinases with a similarly situated cysteine
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residue, selectivity can be explained by examination of the primary structure. The
tyrosine kinase BMX, whose sequence identity is the most similar to that of BTK,
presented an insurmountable challenge in achieving selectivity (Table S2 in the
Supporting information). The Tec kinase ITK also shares a high degree of homology to
BTK, but contains a bulky gatekeeper phenylalanine residue, which explains the higher
observed selectivity of evobrutinib for BTK over ITK. Conversely, other cysteine
containing kinases that are not inhibited by evobrutinib diverge more structurally from
BTK, BMX, and ITK (Figure S5 in the Supporting information).
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Evobrutinib’s selectivity for BTK over EGFR is explained by analysis of the global
differences between the structures of the two cysteine-containing kinases. Upon binding
to BTK, evobrutinib and ibrutinib resulted in a relatively small displacement of N- and C-
lobes in relation to one another. On the other hand, EGFR showed a dramatic
rearrangement in the N-lobe when targeted by ibrutinib (Figure S5 in the Supporting
Information). It is hypothesized that the 1,3-disubstituted pattern on the piperidine ring in
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ibrutinib allows the warhead to rotate to adapt to kinases that move their N lobes. In
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contrast, the evobrutinib warhead is more constrained having a 1,4-substitution on the
piperidine linker and thus cannot accommodate kinases that upon binding rearrange
their N lobe, such as EGFR. This is demonstrated in Table 5, which shows that
selectivity for BTK over EGFR was lost in A14 which, similar to ibrutinib, had a 1,3
attachment to the piperidine ring.
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Table 5. Selectivity of A14 and Evobrutinib for BTK and EGFR
Compound
Structure
BTK IC50 (nM)
EGFR IC50 (nM)
A14
6.5
550
A18
8.9
5800
Evobrutinib
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