Discovery of potent and selective reversible Bruton's tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2021.116163

Bruton's tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase member of the TEC family of tyrosine kinases. Pre-clinical and clinical data have shown that targeting BTK can be used for the treatment for B-cell disorders. Here we disclose t

Full text of DOI:10.1016/j.bmc.2021.116163

Bioorg. Med. Chem. 40 (2021) 116163
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent and selective reversible Bruton’s tyrosine
kinase inhibitors
,
Hui Qiua *, Zahid Alia, Andrew Bendera, Richard Caldwella, Yi-Ying Chenb, Zhizhou Fangc,
Anna Gardbergd, Nina Glaserc, Anja Goettschec, Andreas Goutopoulosa, Roland Grenningloha,
Bettina Hanschkec, Jared Heada, Theresa Johnsona, Christopher Jonesa, Reinaldo Jonesa,
Shashank Kulkarnia, Christine Maurerc, Federica Morandie, Constantin Neagua,
Sven Poetzschc, Justin Potnicka, Ralf Schmidta, Katherine Roea, Ariele Viacava Follisa,
Carolyn Winga, Xiaohua Zhua, Brian Sherera
a EMD Serono Research & Development Institute, 45A Middlesex Turnpike, Billerica, MA 01821, USA1
b Stoke Therapeutics, 45 Wiggins Ave, Bedford, MA 01730, USA
c Merck KGaA, Frankfurter Strasse 250, Darmstadt, Hessen, DE 64293, Germany
d Constellation Pharmaceuticals, 215 First St #200, Cambridge, MA 02142, USA
e Roche Pharma Research and Early Development, Grenzacherstrasse 124, Basel, Basel-Stadt, CH 4070, Switzerland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase member of the TEC family of
tyrosine kinases. Pre-clinical and clinical data have shown that targeting BTK can be used for the treatment for B-
cell disorders. Here we disclose the discovery of a novel imidazo[4,5-b]pyridine series of potent, selective
reversible BTK inhibitors through a rational design approach. From a starting hit molecule 1, medicinal chem-
istry optimization led to the development of a lead compound 30, which exhibited 58 nM BTK inhibitory potency
in human whole blood and high kinome selectivity. Additionally, the compound demonstrated favorable phar-
macokinetics (PK), and showed potent dose-dependent efficacy in a rat CIA model.
Reversible BTK inhibitor
Bruton’s tyrosine kinase
Selective
H3 selectivity pocket
1. Introduction
agammaglobulinemia (XLA), resulting from mutations in Btk, can lead
to B cell specific immunodeficiency. B-cell development in those XLA
Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyro-
sine kinase belonging to the TEC family of tyrosine kinases. It is
expressed in B cells, macrophages, neutrophils, and monocytes, but not
T cells.1 BTK plays a crucial role in signaling through the B-cell antigen
patients is almost completely blocked at the pro-B-cell to pre-B-cell
transition leading to extremely low to undetectable levels of mature B
cells and, in turn, immunoglobulins.3 All the aforementioned findings
provided important evidence that inhibition of BTK is a promising
approach for the treatment for B-cell disorders.4,5
receptor (BCR) in B cells and the Fcγ/Fcε receptors in myeloid cells.
Spontaneous mutations in the pleckstrin homology domain of the Btk
gene found in X-chromosome-linked immune-deficiency (xid) mice and
targeted mutations in Btk knockout mice both result in B-cell develop-
ment and proliferation defects, such as reduced numbers of mature
circulating B cells, greatly reduced serum titers of certain immuno-
globulin isotypes and poor T-independent antibody responses.2 X-linked
Consequently, researchers have expressed considerable interest in
the development of small-molecule BTK inhibitors in recent years. And
multiple reversible and covalent irreversible BTK inhibitors have been
discovered.6–17 Compared to the traditional reversible BTK inhibitors,
covalent irreversible inhibitors targeting Cys481 in the ATP binding
pocket possess several unique advantages, such as high potency,
Abbreviations: ACN, acetonitrile; BTK, Bruton’s tyrosine kinase; CIA, collagen-induced arthritis; DCM, dichloromethane; dppf, 1,1′-bis(diphenylphosphino)
ferrocene; Fsp3, the fraction of sp3 carbon atoms; PBMC, peripheral blood mononuclear cell; PK, pharmacokinetics; QD, once a day; SAR, structure-activity rela-
tionship; SFC, super fluid chromatography; TEA, triethylamine.
* Corresponding author.
E-mail address: hui.qiu@emdserono.com (H. Qiu).
1
A business of Merck KGaA, Darmstadt, Germany.
https://doi.org/10.1016/j.bmc.2021.116163
Received 7 February 2021; Received in revised form 6 April 2021; Accepted 11 April 2021
Available online 20 April 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
extended target occupancy, and feasibility to measure the occupancy via
probe molecules. To date two generations of small molecule covalent
irreversible BTK inhibitors have been used in the treatment of B-cell
malignancies, and demonstrated clinical efficacy in previously difficult
to treat diseases.15,18,19,20 It is worth mentioning that to circumvent the
antibody-dependent cell-mediated cytotoxicity of the 1st generation
first-in-class BTK covalent irreversible inhibitor ibrutinib,8 the 2nd
generation molecules, such as acalabrutinib,8,21 evobrutinib,15 zanu-
brutinib,22 displayed more refined pharmacological profiles and
demonstrated a more favorable safety profile. However, the possible
toxicity concern related to the inhibition of cysteine in other kinases
with the warheads of hose covalent molecules can’t be ignored. Espe-
cially 10 kinases (BMX, TEC, ITK, TXK, EGFR, ERBB2, ERBB4, JAK3,
BLK and MKK7) also possess a cysteine in the same area as Cys481 in
BTK.23 Moreover, the reactive warhead in some molecules might also
react with other nucleophiles besides cysteine.6 Finally, clinical resis-
tance of several cancers resulting from mutations of Cys481 have
already been observed, which could diminished the advantage of co-
valent molecules.24 To overcome those liabilities related to covalent
inhibitors, a new generation of reversible BTK inhibitors occupying the
H3 selectivity pocket25 in an inactive conformation of BTK has been
developed.17,26 The most advanced example is clinical candidate GDC-
0853 (fenebrutinib). It displayed excellent kinome selectivity, inhibit-
ing only 3 of 286 off-target kinases. On the basis of the determined IC50
values, the selectivity for BTK was > 100-fold against each of 3 notable
off-targets: BMX (153-fold), FGR (168-fold), and SRC (131-fold).6,27,28
Very recently, a new covalent irreversible BTK inhibitor LOU064
(remibrutinib) was disclosed, which was claimed to have a better
kinome selectivity than several other covalent irreversible molecules.
It’s worth noting that the excellent kinome selectivity was largely
attributed to the molecule occupying the H3 selectivity pocket.29
In an effort to produce more advanced BTK inhibitors, we have
worked to refine the designs of past molecules. Herein, we describe the
design, synthesis and pharmacological evaluation of a novel series of
imidazo[4,5-b]pyridine series of reversible BTK inhibitors occupying the
BTK H3 selectivity pocket. The lead molecule 30, which was discovered
via optimization of hit molecule 1, is a potent, selective BTK inhibitor
with good preclinical PK profile and shows potent efficacy in the rat CIA
model of rheumatoid arthritis.
consists of Phe413 (P-loop), Leu542, Val546 and Tyr551 (activation
loop) (Fig. 1).25,30 Occupying the H3 selectivity pocket has been shown
to be a successful strategy in achieving high kinome selectivity in several
non-covalent reversible BTK inhibitors, including RN-486, CGI-1746,
GDC-0834 and GDC-0853.6,16,17a Inspired by those works, our initial
efforts were focused on design of potent molecules that occupy the H3
selectivity pocket of BTK and sequester Tyr551. Based on a rational
design approach, we discovered hit compound 1. As a good starting
point, compound 1 showed encouraging single-digit nM enzymatic po-
tency. To better understand the ligand-receptor interactions, we ob-
tained the X-ray crystal structure of compound 1 with BTK (Fig. 1, PDB
code: 7KXM). Confirming our hypothesis, the t-butyl phenyl amide
group approaches Tyr551 of the H3 selectivity pocket. The carbonyl of
amide forms a hydrogen-bond with Lys430. We observed that the salt
bridge between Lys430 and Glu445, which exists in the BTK active
conformation is broken.31 The imidazo[4,5-b]pyridine core lies in
proximity of the hinge area interacting with residues Met477. The
carbonyl morpholine moiety extends into the open solvent accessible
front pocket (FP).
2.2. H3 pocket group exploration
Despite the good enzymatic potency, several other parameters of
compound 1 still needed to be optimized. For example, it showed very
low A-B permeability (Papp A-B = 0.02 × 10ꢀ 6 cm/s) and extremely high
efflux ratio (ER = 1150) in a Caco-2 assay test. It’s known that poor
permeability could lead to low exposure in following PK studies.32
Consequently, one important task in hit optimization was mitigation of
the permeability liability. Due to the existence of multiple aromatic
`
rings in the scaffold, the human ether-a-go-go-related gene (hERG)
Kv11.1 channel inhibitory activity was also closely monitored. Selected
representatives are included in Table 1.
Reduction of topological polar surface area (tPSA) has been suc-
cessfully applied in our former research to improve permeability and
reduce ER.33,34 Based on our experience from former SAR, we planned to
reduce tPSA (100.2 Å2) of molecule 1 via removal of the heteroatoms in
the front pocket group (FPG). One potential advantage of this strategy is
that we might manipulate tPSA of newly designed compounds without
disrupting the interactions between the kinase and rest of the ligand.
With this strategy in mind, compound 2 (tPSA = 88.5 Å2) was finally
discovered with deletion of the amide moiety and replacement of the
remaining phenyl group with more soluble methyl substituted pyrazole.
This newly designed compound 2 showed a dramatically reduced efflux
ratio (ER = 42 vs 1150 in compound 1), suggesting a path forward. In
fact, methyl pyrazole was also used by other researchers as the front
pocket group (FPG) in their top molecules.17b,c To avoid possible toxicity
issues associated with the aniline moiety in 2, one methylene group was
introduced into the scaffold and the t-butyl phenyl amide was trans-
positioned to C4 of the phenyl link accordingly. After several design
iterations, compound 3 was discovered and displayed more than a 10-
fold boost in potency without undermining the acceptable perme-
ability. We reasoned that the improved potency might result from the
newly added methylene group that provided the optimal vector for the
phenyl amide moiety occupying the H3 pocket. To expand the SAR, we
also explored the replacement of the methyl substituent on the phenyl
ring with other groups, for example fluorine, because it has been re-
ported that introducing a fluorine into the molecule may have a positive
effect on increasing the permeability.35 Moreover, we hypothesized that
the installed fluorine might form an intramolecular hydrogen bond with
the proximal amide NH group, which would decrease hydrogen bond
donor (HBD) number and efflux ratio.36 Unfortunately, this replacement
in compound 4 didn’t lead to any meaningful change in permeability
and resulted in a 5-fold loss of potency. To further expand the SAR and
lower the high lipophilicity, the distal phenyl
2. Results and discussion
2.1. Discovery of novel imidazopyridine series of reversible BTK inhibitors
Our goal was to identify a potent and selective BTK inhibitor with
efficacy in models of autoimmune diseases while demonstrating an
excellent safety profile. A high kinome selectivity is essential to achieve
this goal.
The H3 selectivity pocket is a hydrophobic pocket in BTK that
group in compound 4 was replaced with hetero aromatic rings,
which led to improved permeability in compound 5 along with a 6-fold
Fig. 1. X-ray crystal structure of compound 1 bound to BTK (PDB code: 7KXM).
2

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
Table 1
Table 1 (continued)
Compound
The H3 pocket group exploration.
BTK
Caco2 A-
B (×10ꢀ 6
cm/s)/ER
clogP
hERG
enzymatic/
hPBMC IC50
[nM]a
inhibitory
Compound
BTK
Caco2 A-
B (×10ꢀ 6
cm/s)/ER
clogP
4.5
hERG
activity
enzymatic/
hPBMC IC50
[nM]a
inhibitory
b
(IC50
)
activity
b
(IC50
)
1
2
3
5.1/1680
0.02/
1150
ꢀ 24%
ꢀ 18%
ꢀ 42%
16/–
0.6/62
0.2/44
3.8
4.1
10
11
27/–
–
3.8
3.6
–
–
1.3/54
1.5/820
0.3/15
4
5
6
6.2/151
0.4/24
11/4.8
2.8/41
4.1
3.1
2.5
ꢀ 18%
ꢀ 44%
ꢀ 44%
12
13
63/–
–
2.6
3.9
–
0.4/26(41)c
1.9/64
0.2/6
ꢀ 67% (2.6
μ
M)
0.4/118
14
15/–
–
3.8
–
7
8
0.4/14
0.5/77
2.5/30
3/20
3.1
3.3
–22%
15
16
28/–
8/141
3.7
2.8
–
ꢀ 35%
1/256
0.4/95
ꢀ 1%
9
0.5/211
0.6/8
4.6
–33%
a
IC50 values are shown as mean values of at least two determinations unless
specified otherwise.
b
Percentage inhibition at 10 μM.
3

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
c
Human whole blood IC50
.
substantial potency drop. In the end, the exploration of the front pocket
group didn’t lead to a molecule with superior properties over compound
5.
boost in potency. Analogues 6–9 bearing different five-member hetero
aromatic rings were prepared. But none of them had comparable po-
tency or permeability as compound 5. Analogs 10–13 with six-
membered aromatic rings were also tested. All of them were notice-
ably less potent with the exception of lactam 13, which unfortunately
2.4. Addressing poor solubility
However, attempts to progress molecule 5 forward were halted
because of the non-optimal kinetic and thermodynamic solubility of the
molecule (kinetic solubility = 1.7 µM, Fasted state simulated intestinal
fluid (FaSSIF) solubility = 2 µg/mL). Our concern stemmed from our
experience that compounds with low FaSSIF solubility could have un-
desirable exposure, which would eventually lead to low efficacy.
Consequently, our highest priority in the subsequent optimization was
focused on improvement of FaSSIF solubility while maintaining other
drug-like properties. We speculated the poor solubility of compound 5
might be related to the planar geometry of this molecule, which was
confirmed by the single X-ray crystal structure of compound 5 (Fig. 2).
Moreover, the molecules were heavily packed due to multiple inter-
molecular interactions (shown with blue dash lines in Fig. 2). We
envisioned that introduction of an intra-molecular clash to destroy the
planar geometry of this scaffold could interrupt the intermolecular in-
teractions and consequently improve the solubility.37 Because the
phenyl linker is essential for potency, one of our strategies to address the
poor FaSSIF solubility was to install substituent at the C6 position of the
phenyl linker (shown with red arrow in Fig. 2) to increase of the dihedral
angle between the phenyl linker and the pyridine core.
showed a strong hERG inhibition (IC50 = 2.6 μM). Introduction of a
saturated ring in compounds 14–16 were also screened but again turned
out to be fruitless. We reasoned the loss of potency in 14 and 15 might be
due to the collision between the saturated rings in them and the re-
ceptor. Although incorporation of a smaller azetidine ether moiety in
compound 16 regained partial potency, it displayed an unacceptable
high efflux ratio of 95. Having the most balanced profiles, compound 5
was chosen to move forward as the most advanced molecule.
2.3. Front pocket group exploration
We also explored different front pocket groups (FPG) using com-
pound 5 as the model molecule. Several selected examples were listed in
Table 2. Considering FPG extend into the solvent exposed front pocket,
solubilizing groups were introduced into compound 17 and 18. Both
molecules maintained potency similar to compound 5 but showed
higher efflux ratio, which might relate to the increased tPSA with the
addition of oxygen and nitrogen atoms. Replacement of pyrazole with
pyridine resulted in low efflux ratio (ER = 1.3) in compound 19. How-
ever, it didn’t show satisfying potency in the following cellular activity
test. Introduction of a small cyclopropyl in compound 20 led to a
To evaluate this hypothesis and generate new design ideas, we ob-
tained the X-ray crystal structure for 5 bound with BTK (Fig. 3, PDB ID:
7KXL). Analysis of the crystal structure indicates compound 5 adopts a
similar binding mode as compound 1. The imidazopyridine core in-
teracts with residues Glu475 and Met477 as the hinge binder. The
methyl pyrazole front pocket group (FPG) exposes to the open and
flexible solvent pocket.38,39 The fluorine atom in the linker points to the
ribose pocket while the amide bond extends into the H3 selectivity
pocket with an opposite vector. Glu445, which forms the conserved salt
bridge with Lys430 in the BTK active confirmation, is pushed away.
Instead Asp539 gets close to Lys430. The t-butyl oxadiazole group oc-
cupies the H3 selectivity pocket with the t-butyl group insertion into the
lipophilic pocket engaging Tyr551. We did observe that the receptor
binding pocket around the C6 position of the phenyl linker should
accommodate a small group. Accordingly, several molecules bearing
small substituents at this position were synthesized and tested. How-
ever, those newly designed molecules didn’t show superior profiles to
compound 5 (for example, FaSSIF solubility) and ultimately, they were
not chosen for further characterization. (Data not shown).
Table 2
Front pocket group exploration.
Compound
BTK
Caco2
A-B
clogP
2.4
hERG
enzymatic/
hPBMC IC50
[nM]a
inhibitory
(×10ꢀ 6
cm/s)/
ER
activityb
17
18
19
20
0.7/23
0.3/63
1.2/251
12/–
0.8/44
0.5/145
2.2/1.3
–
ꢀ 41%
2.6
3.6
3.0
–
2.5. Linker optimization effort
With the unexpected failure of linker modification on C6 position, we
re-evaluated the X-ray crystal structure of 5 and noticed that the pocket
around the distal methylene group might tolerate larger substituents
than hydrogen (shown with red arrow in Fig. 3). We speculated the
introduction of sp3 carbon-containing groups to occupy this region could
ꢀ 17%
–
a
b
IC50 values are shown as mean values of at least two determinations.
Percentage inhibition at 10
μ
M.
Fig. 2. Crystal structure of compound 5.
4

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
<0.3%/0.43%). The intrinsic clearances in human/mouse/rat micro-
some were medium to high (Human/mouse/rat microsomal CLint = 32/
75/67 (
μ
L*minꢀ 1 mgꢀ 1)). Compound 30 was stable in human or mouse
plasma (>85% remaining in 4 h). It is mainly metabolized by CYP3A4
but not by aldehyde oxidase (AOX). Moreover, it is an inhibitor of
CYP2C9 and CYP2C19 with IC50 value of 3.7
respectively.
μM and 4.1 μM,
Selectivity profile of 30
30 was tested at 1 μM against a broad biochemical panel containing
more than 380 human kinases along with RN486. Like other BTK in-
hibitors occupying the H3 selectivity pocket, 30 shows excellent kinome
selectivity, inhibiting 2 of the off-target kinase (Src, FGR) with activity
in range of the on-target inhibition of BTK (>90% inhibition). Single
point inhibition data prompted follow up dose–response screening.
Targets with > 80% percentage inhibition by 30 and RN486 were listed
in Table 4 (Table S2A and S2B in Supplementary data). Although both
compounds inhibit BTK potently, the off-target profiles are not quite the
same.
Fig. 3. X-ray crystal structure of compound 5 bound to BTK (PDB code: 7KXL).
X-ray crystal structure of 30
not only interrupt the aforementioned intermolecular interaction be-
tween proximal amide NH and pyrazole front pocket group, but also
increase Fsp3.40 Both of these changes should help improve the solubi-
lity. Confirming our speculation, compound 21 with cyclopropyl sub-
stituent showed 25-fold boost in FaSSIF solubility (56 µg/mL for 21 vs 2
µg/mL for 5) (Table 3). Encouraged by this result, we prepared more
analogs, including 22 with an sp3 carbon-containing bicyclic linker.
Pleasantly, distomer 22A, although less potent than 5, demon-
strated>90-fold improved FaSSIF solubility. The eutomer was aban-
doned due to strong hERG inhibition and worse permeability.
To confirm the absolute configuration of 30 and understand the
ligand-receptor binding information, we obtained the X-ray crystal
structure of 30 bound with BTK (Fig. 4, PDB code: 7KXQ). The binding
mode is similar to compound 1 and 5. It has the expected interactions
between the imidazo[4,5-b]pyridine core and residue Met477 in the
hinge area. The seven-member ring moiety may help lock the favorite
conformation, facilitating the t-butyl oxadiazole amide group occupying
the H3 selectivity pocket and approaching Tyr551. Same as what we
observed in Fig. 1 and Fig. 3, the oxygen atom of the amide bond in-
teracts with Lys430. The conserved salt bridge between Lys430 and
Glu445 in BTK active confirmation is broken. The tri-substituted pyr-
azole front pocket group is exposed to the solvent pocket. It’s clear that
compound 30 adopted an S conformation.
2.6. Optimization of compound 22A
Rat PK study of 30
Our subsequent effort aimed to further improve the potency of 22A
(Table 3). The established SAR demonstrated that compound 17 with a
tetrahydropyran (THP) group showed similar potency as compound 5.
Following this lead, 23 with a THP containing a front pocket group was
prepared. Encouragingly, 23 did show a boosted potency, albeit with an
increased efflux ratio (ER = 129), which could be related to the
increased tPSA. To address the high efflux ratio of 23, we introduce
steric hindrance to compound 24 and 25 via decorating the pyrazole
with alkyl groups.34 We speculated the increased steric hindrance
should also interrupt the pyrazole-involved intermolecular interactions
shown in Fig. 2 and increase Fsp3 of the molecules. As expected, we
observed lower efflux ratio in 24 and 25. To further expand the SAR,
analogues 26–29 with more alkyl group additions around pyrazole were
synthesized. Among them, compound 28 displayed an encouraging IC50
of 25 nM in hPBMC assay. More excitingly, the increased steric hin-
drance at the front pocket group region in 28 also brought in good
FaSSIF solubility (56 µg/mL), which is more than 25-fold higher than 5.
The steric hindrance in 28 might also contribute to the weak hERG in-
hibition (-18% inhibition). Removal of methyl decorations from com-
pound 28 led to noticeable drop of potency, FaSSIF solubility and
stronger hERG inhibition with 29, which supported the essential role of
methyl substitution.
Because of its promising in vitro profile, compound 30 was tested in
rat pharmacokinetic studies (Table 5). At 10 mg/kg PO, 30 showed
bioavailability of 77%.
Preclinical Efficacy in Rheumatoid Arthritis
Due to promising in vitro profiles and in vivo PK results, molecule 30
was tested for efficacy in a rat collagen-induced arthritis (CIA) model
along with RN486. Female Lewis rats immunized with collagen were
dosed orally (day 6–16) with 30 (2, 10 and 50 mg/kg QD) or RN486 (30
mg/kg QD) (n = 10/group) (Fig. 5A). Animals were terminated on study
day 17. Disease-induced ankle-swelling was significantly reduced for
rats treated with 30 at 10 mg/kg and 50 mg/kg, compared with vehicles.
Better efficacy was observed with 50 mg/kg of 30 than 30 mg/kg of
RN486. After dosing at day 16, the plasma concentration of compound
30 in the plasma was measured at different timepoints. It was found that
oral dosing with 30 at 50 mg/kg maintained plasma concentrations
above the rat whole blood BTK IC50 (black dotted line, 11 nM or 6.3 ng/
mL) for 24 h, while the 10 mg/kg dose maintained plasma concentration
above the IC50 for a minimum of 16 h of the 24 h dosing period (Fig. 5B).
Synthesis of compound 30
The preparation of top compound 30 started with polyphosphoric
acid catalyzed condensation of 31 and carboxylic acid 32 to generate the
intermediate 33 (Scheme 1). Coupling 33 with boronic ester 34 under
Suzuki reaction condition afforded the Boc-protected 35. HCl mediated
deprotection and an amide coupling reaction catalyzed by propane-
phosphonic acid anhydride (T3P) provided the racemic mixture, which
was separated via chiral SFC to give compound 30. The synthesis of
compound 1–29 can be found in Supplementary data.
Encouraged by the promising profile of 28, molecule 30 with a more
flexible seven-member was also prepared, which exhibited comparable
IC50 in hPBMC and human whole blood (hWB) assay to RN486. Further
profiling of this molecule showed low hERG inhibition and good FaSSIF
solubility. In the end, compound 28 was moved forward due to the
balanced profile along with the most potent compound 30. However,
compound 28 displayed an inferior efficacy profile in rat CIA model
study (Data not shown).
3. Conclusions
Profiles of compound 30
As the top molecule, 30 was further characterized in different in vitro
assays. Protein binding test revealed a low unbound fraction of the drug
across rodent species and humans (fu, human/mouse/rat = <0.3%/
In summary, we have discovered a potent and selective reversible
BTK inhibitor 30 bearing a imidazo[4,5-b]pyridine scaffold. Guided by
X-ray crystal structure of 1 bound with BTK and via a strategy of
5

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
Table 3
Optimization of compound 22A.
Compounda
d
FaSSIF (µg/mL)
BTK enzyme/hPBMC IC50 [nM]b
2.7/36
Caco2 A-B (×10ꢀ 6 cm/s)/ERc
hERG inhibitory activity (IC50)
21
56
1.1/80
ꢀ 41%
22
–
0.7/54
1.9/98
1.0/59
5/4.8
ꢀ 46%
–23%
22Ae
190
22Be
–
–
1.1/39
1.2/51
1.0/49
ꢀ 55% (6.3 μM)
23
0.4/129
–32%
24f
7
0.5/30
0.9/87
5/3
ꢀ 17%
–32%
25f
33
1.3/20
26f
28
1/137
0.6/4
2/4.3
ꢀ 31%
ꢀ 46%
27e
7
1.1/62
(continued on next page)
6

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
Table 3 (continued)
Compounda
d
FaSSIF (µg/mL)
BTK enzyme/hPBMC IC50 [nM]b
Caco2 A-B (×10ꢀ 6 cm/s)/ERc
hERG inhibitory activity (IC50)
28e
56
25
0.6/25(92)g
3.4/1.8
2.6/3
ꢀ 18%
ꢀ 35%
29e
0.23/54
30f
33
31
0.4/3(58)g
0.7/4.5
4.6/5.7
ꢀ 42%i
RN486
0.9/39(26)g
ꢀ 62% (1.4 µM)
a
Between two enantiomers obtained from chiral SFC separation, only the enantiomer with overall superior profiles, such as better permeability, potency, solubility
and lower hERG inhibition were included for SAR discussion.
b
IC50 values are shown as mean values of at least two determinations unless specified otherwise.
c
d
Similar SAR was observed using passive permeability data (Table S1 in Supplementary data).
Percentage inhibition at 10 μM.
e
f
Absolute stereochemistry is not known.
Absolute stereochemistry is confirmed by X-ray crystal structures (Supplementary data).
g
i
Human whole blood IC50
.
Percentage inhibition at 30 μM.
Table 4
Selectivity profile of 30 and RN486.
IC50(nM)
BTK
FGR
SRC
Aurora-A
MINK1
BMX-ETK
TEC
YES1
30
4.15
2.25
5.36
180
27.12
60.08
90.73
–
–
5.69
–
RN486
–
–
–
62.04
1592
lowering tPSA and lipophilicity, our optimization led to the discovery of
molecule 5 with improved potency and permeability profiles. To miti-
gate the solubility issue of compound 5, we conducted small molecule X-
ray crystal analysis, which revealed the planar geometry and heavily
packed crystal form with multiple intermolecular interactions. We
speculated that breaking the planar geometry via installation of sp3
carbon-containing moiety should help improve the solubility. The effort
ultimately resulted in the discovery of compound 30 showing a 30-fold
improvement in FaSSIF solubility compared to hit molecule 1. Com-
pound 30 displayed excellent kinome selectivity, only inhibited two off-
target kinases FGR and Src with closest inhibitory activity as BTK. This
can be attributed to the binding mode of 30, in which it occupies the H3
selectivity pocket of an inactive conformation of BTK. Finally compound
30 showed promising rat PK profile and good efficacy with 50 mg/kg QD
dose without major safety signals.
4. Experimental part
Material and methods
All the reagents and solvents were commercially available. 1H and
C13 NMR experiments were recorded on a Varian 500 MHz VNMRS
spectrometer equipped with a Varian One NMR probe, or a Bruker
Avance III 400 MHz spectrometer equipped with a Bruker 400 BBO
probe. Chemical shifts are expressed in δ ppm with TMS as an internal
standard (δ = 0 ppm). Abbreviations used in describing peak signals are
br = broad signal, s = singlet, d = doublet, dd = doublet of doublets, t =
triplet, q = quartet, m = multiplet. All microwave reactions were con-
ducted using the Biotage Initiator. All hydrogenation reactions were
7

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
conducted using the H-Cube apparatus manufactured by ThalesNano.
All final compounds were determined to have purity > 95% by HPLC.
The purity was assessed using an Agilent 1100 HPLC system (Agilent
Technologies) with an XBridge column (C18, 3.5 μm, 4.6 mm × 50 mm).
Mass spectra were recorded on an electrospray mass spectrometer (ESI-
MS) in electrospray ionization positive (ESI+) mode. High-resolution
mass spectrometry (HRMS) experiments were performed on a CapLC
(1100 series, Agilent Technologies) which was coupled to the Synapt G1
HDMS (Waters, Milford, MA) mass spectrometer using ESI as the ioni-
zation source. Chiral SFC separation was performed with Waters SFC
system (model number–SFC P200).
Preparation of compound 30
4.0.1. 7-Bromo-2-(1-isopropyl-3,5-dimethyl-1H-pyrazol-4-yl)–3H-
imidazo[4,5-b]pyridine (33)
Fig. 4. X-ray crystal structure of compound 30 bound to BTK (PDB
A reaction vial containing 4-bromopyridine-2,3-diamine 31 (400.00
mg; 2.13 mmol; 1.00 eq.), 1-isopropyl-3,5-dimethyl-1H-pyrazole-4-car-
boxylic acid 32 (407.03 mg; 2.23 mmol) and polyphosphoric acid (4.00
g) was heated at 100 ◦C overnight. Saturated ammonium chloride so-
lution was added into the reaction mixture, and the pH was adjusted to 7
with NaOH pellets. The product precipitated and was dried after filtra-
tion. The solid was directly used in the next step. [ESI-MS] (ESI+): m/z
calcd for C14H17BrN5 [M+H]+ 334.07; found 334.00.
code: 7KXQ).
Table 5
in vivo Rat Pharmacokinetic parameters of 30.
PO 10 mg/kga
AUC0-6h (ng * h/L) Cmax (ng/mL) F (%)c t1/2 (h)
3030 849 77 1.93
IV 0.2 mg/kgb
AUC0-4h (ng * h/L) CL (L/h/kg) Vss (L/kg)
79 2.42 2.62
{2-[2-(1-Isopropyl-3,5-dimethyl-1H-pyrazol-4-yl)–3H-imidazo[4,5-
b]pyridin-7-yl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl}-carba-
mic acid tert-butyl ester (35)
a
b
c
Average of two animals. Vehicle: PEG 300 / NMP (90/10).
Average of three animals. Vehicle: DMSO (1%)/PEG200 (40%) in water.
Based on AUC of 0–6 h.
To a 5 mL microwave vial containing [2-(4,4,5,5-tetramethyl-[1,3,2]
dioxaborolan-2-yl)-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl]-car-
bamic acid tert-butyl ester 34 (92.71 mg; 0.24 mmol), 7-bromo-2-(1-
isopropyl-3,5-dimethyl-1H-pyrazol-4-yl)–3H-imidazo[4,5-b]pyridine
33 (80.00 mg; 0.24 mmol), [1,1′-bis(diphenylphosphino)ferrocene]
dichloropalladium (ii), complex with dichloromethane (29.32 mg; 0.04
mmol) and sodium carbonate (55.81 mg; 0.53 mmol) were added
ethanol (6 mL) and water (0.60 mL). The mixture was evacuated and
backfilled with nitrogen before it was stirred at 115 ◦C for 16 h before it
was purified with basic pre-HPLC (0.1% NH4OH-H2O/ACN). The
desired fractions were dried to give the title product as a solid (60 mg,
yield 48.7%). [ESI-MS] (ESI+): m/z calcd for C30H39N6O2 [M+H]+
515.31; found 515.20. 1H NMR (400 MHz, DMSO‑d6) δ 12.73 (s, 1H),
8.28 (d, J = 5.2 Hz, 1H), 8.18 (d, J = 7.2 Hz, 2H), 7.66–7.44 (m, 2H),
7.34 (d, J = 8.1 Hz, 1H), 4.78 (t, J = 9.1 Hz, 1H), 4.58 (p, J = 6.6 Hz,
1H), 2.92 (s, 2H), 2.56 (s, 3H), 2.43 (s, 3H), 1.96–1.68 (m, 4H), 1.56 (m,
1H), 1.49–1.22 (m, 16H).
2-[2-(1-Isopropyl-3,5-dimethyl-1H-pyrazol-4-yl)–3H-imidazo[4,5-b]
pyridin-7-yl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ylamine hy-
drochloride (36)
To a reaction vial containing {2-[2-(1-isopropyl-3,5-dimethyl-1H-
pyrazol-4-yl)–3H-imidazo[4,5-b]pyridin-7-yl]-6,7,8,9-tetrahydro-5H-
benzocyclohepten-5-yl}-carbamic acid tert-butyl ester 35 (60.00 mg;
0.12 mmol) in methanol (10.00 mL) was added hydrogen chloride 4.0 N
in dioxane (0.30 mL; 1.2 mmol). The reaction was stirred at rt for 16 h
before it was concentrated and used in the next step directly. [ESI-MS]
(ESI+): m/z calcd for C25H31N6 [M+H]+ 415.26; found 415.30.
3-tert-Butyl-[1,2,4]oxadiazole-5-carboxylic acid {2-[2-(1-isopropyl-
3,5-dimethyl-1H-pyrazol-4-yl)–3H-imidazo[4,5-b]pyridin-7-yl]-6,7,8,9-
tetrahydro-5H-benzocyclohepten-5-yl}-amide (30)
To a reaction vial containing 2-[2-(1-isopropyl-3,5-dimethyl-1H-
pyrazol-4-yl)–3H-imidazo[4,5-b]pyridin-7-yl]-6,7,8,9-tetrahydro-5H-
benzocyclohepten-5-ylamine hydrochloride 36 (55.00 mg; 0.12 mmol)
and 3-tert-butyl-1,2,4-oxadiazole-5-carboxylic acid 37 (20.75 mg; 0.12
mmol) in dichloromethane (4.00 mL) was added triethyl amine (0.17
mL; 1.22 mmol). After the mixture was stirred at rt for 5 min, 2,4,6-tri-
propyl-[1,3,5,2,4,6]trioxatriphosphinane 2,4,6-trioxide (38.80 mg;
0.12 mmol) was slowly added into the mixture. The reaction mixture
was stirred for 30 min before it was concentrated and purified by basic
Fig. 5. Efficacy of compound 30 in rat CIA model. (A) Female Lewis rats with
develping CIA were dosed orally (day 6–16) with vehicle, the indicaded doses
of 30 and RN486. Daily ankle diameter measurements are shown as mean ±
SEM and were reduced toward normal for all drug-treated rats as compared
with the respective disease control. (B) Total plasma concentration of 30 at day
16 after dosing (2, 10, 50 mg/kg). The dot line denotes the rat whole blood BTK
IC50 = 11 nM. Statistical significance determined by ANOVA.
8

H. Qiu et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116163
Scheme 1. Synthetic route of compound 30. Reagents and conditions: (a) polyphosphoric acid (PPA), 100 ◦C, 12 h; (b) Pd(dppf)2Cl2∙DCM, Na2CO3, ethanol/H2O
(10:1 v/v), 115 ◦C, 16 h; (c) HCl, MeOH; (d) TEA, T3P, DCM, 0.5 h, followed by chiral SFC separation.
development. J Med Chem. 2018;61:2227–2245. https://doi.org/10.1021/acs.
pre-HPLC (0.1% NH4OH-H2O/ACN). The desired fractions were dried to
jmedchem.7b01712.
afford the title product as a white solid (20 mg, HPLC purity 99%, yield
7
8
Brown JR. Ibrutinib (PCI-32765), the first BTK (Bruton’s tyrosine kinase) inhibitor in
clinical trials. Curr Hematol Malig Rep. 2013;8:1–6. https://doi.org/10.1007/s11899-
012-0147-9.
28.9%). The following chiral SFC separation provided the title com-
pound as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ 12.74 (s, 1H),
9.96 (d, J = 8.1 Hz, 1H), 8.31–8.19 (m, 2H), 8.15 (d, J = 8.0 Hz, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 5.26 (s, 1H), 4.55 (p, J
= 6.7 Hz, 1H), 2.95 (s, 2H), 2.54 (s, 3H), 2.41 (s, 3H), 1.95 (s, 3H), 1.80
(d, J = 10.4 Hz, 2H), 1.43–1.34 (m, 16H). 13C NMR (101 MHz,
DMSO‑d6) δ 178.12, 169.37, 153.08, 150.60, 149.36, 146.07, 143.28,
142.49, 141.33, 138.96, 135.83, 134.78, 133.35, 130.49, 126.88,
124.89, 115.20, 108.97, 53.28, 49.31, 35.70, 34.46, 32.79, 29.32,
28.61, 27.43, 22.78, 13.83, 10.56. [ESI-MS] (ESI+): m/z calcd for
Barf T, Covey T, Izumi R, et al. 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. https://doi.org/10.1124/
jpet.117.242909.
9
Schafer PH, Kivitz AJ, Ma J, et al. Spebrutinib (CC-292) affects markers of B cell
activation, chemotaxis, and osteoclasts in patients with rheumatoid arthritis: results
from a mechanistic study. Rheumatol Ther. 2020;7:101–119. https://doi.org/
10.1007/s40744-019-00182-7.
10 Park JK, Byun JY, Park JA, et al. HM71224, a novel Bruton’s tyrosine kinase
inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a
mouse model: a potential drug for rheumatoid arthritis. Arthritis Res Ther. 2016;18:
91. https://doi.org/10.1186/s13075-016-0988-z.
C
32H39N8O2 [M+H]+ 567.32; found 567.50.
11 Lee SK, Xing J, Catlett IM, et al. Safety, pharmacokinetics, and pharmacodynamics of
BMS-986142, a novel reversible BTK inhibitor, in healthy participants. Eur J Clin
Pharmacol. 2017;73:689–698. https://doi.org/10.1007/s00228-017-2226-2.
12 Wu J, Zhang M, Liu D. Bruton tyrosine kinase inhibitor ONO/GS-4059: from bench to
bedside. Oncotarget. 2017;8:7201–7207. https://doi.org/10.18632/
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
13 Zhao X, Xin M, Huang W, et al. Design, synthesis and evaluation of novel 5-phe-
nylpyridin-2(1H)-one derivatives as potent reversible Bruton’s tyrosine kinase
inhibitors. Bioorg Med Chem. 2015;23:348–364. https://doi.org/10.1016/j.
bmc.2014.11.006.
14 Xin M, Zhao X, Huang W, et al. Synthesis and biological evaluation of novel 7-
substituted 3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines as potent Bruton’s
tyrosine kinase (BTK) inhibitors. Bioorg Med Chem. 2015;23:6250–6257. https://doi.
org/10.1016/j.bmc.2015.08.039.
Acknowledgments
The authors would like to thank Dr. Lesley Liu-Bujalski for her
valuable suggestions in the preparation of this manuscript.
15 Caldwell RD, Qiu H, Askew BC, et al. Discovery of evobrutinib: an oral, potent, and
highly selective, covalent Bruton’s tyrosine kinase (BTK) inhibitor for the treatment
of immunological diseases. J Med Chem. 2019;62:7643–7655. https://doi.org/
10.1021/acs.jmedchem.9b00794.
Appendix A. Supplementary data
16 Lou Y, Han X, Kuglstatter A, et al. Structure-based drug design of RN486, a potent
and selective Bruton’s tyrosine kinase (BTK) inhibitor, for the treatment of
rheumatoid arthritis. J Med Chem. 2015;58:512–516. https://doi.org/10.1021/
jm500305p.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116163.
17 (a) Young WB, Barbosa J, Blomgren P, et al. Potent and selective bruton’s tyrosine
kinase inhibitors: discovery of GDC-0834. Bioorg Med Chem Lett. 2015;25:
1333–1337. https://doi.org/10.1016/j.bmcl.2015.01.032.(b) Kawahata W,
Asami T, Kiyoi T, et al. Design and synthesis of novel amino-triazine analogues as
selective Bruton’s tyrosine kinase inhibitors for treatment of rheumatoid arthritis.
J Med Chem. 2018;61:8917–8933. https://doi.org/10.1021/acs.jmedchem.8b01147.
(c) Ma B, Bohnert T, Otipoby KL, et al. Discovery of BIIB068: a selective, potent,
reversible Bruton’s tyrosine kinase inhibitor as an orally efficacious agent for
autoimmune diseases. J Med Chem. 2020;63:12526–12541. https://doi.org/
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