Utilizing structure based drug design and metabolic soft spot identification to optimize the in vitro potency and in vivo pharmacokinetic properties leading to the discovery of novel reversible Bruton's tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2021.116275

Bruton's tyrosine kinase (BTK) is an essential node on the BCR signaling in B cells, which are clinically validated to play a critical role in B-cell lymphomas and various auto-immune diseases such as Multiple Sclerosis (MS), Pemphigus, and rheumatoid arthritis (RA). Although non-selective irreversible BTK inhibitors have been approved for oncology, due to the emergence of drug resistance in B-cell lymphoma associated with covalent inhibitor, there an unmet medical need to identify reversible, selective, potent BTK inhibitor as viable therapeutics for patients. Herein, we describe the identification of Hits and subsequence optimization to improve the physicochemical properties, potency and kinome selectivity leading to the discovery of a novel class of BTK inhibitors. Utilizing Met ID and structure base design inhibitors were synthesized with increased in vivo metabolic stability and oral exposure in rodents suitable for advancing to lead optimization.

Full text of DOI:10.1016/j.bmc.2021.116275

Journal Pre-proofs
Utilizing Structure Based Drug Design and Metabolic Soft Spot Identification
to optimize the in vitro potency and in vivo Pharmacokinetic Properties Lead-
ing to the Discovery of Novel Reversible Bruton’s Tyrosine Kinase Inhibitors
Brian T. Hopkins, Eris Bame, Noah Bell, Tonika Bohnert, Jon K. Bowden-
Verhoek, Minna Bui, Mark T. Cancilla, Patrick Conlon, Patrick Cullen,
Daniel A. Erlanson, Junfa Fan, Tarra Fuchs-Knotts, Stig Hansen, Stacey
Heumann, Tracy J. Jenkins, Chuck Gua, Ying Yiu, YuTing Liu, Mukush
Lulla, Douglas Marcotte, Isaac Marx, Bob McDowell, Elisabeth Mertsching,
Ella Negrou, Michael J. Romanowski, Daniel Scott, Laura Silvian, Wenjin
Yang, Min Zhong
PII:
S0968-0896(21)00283-2
https://doi.org/10.1016/j.bmc.2021.116275
BMC 116275
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 March 2021
4 June 2021
9 June 2021
Please cite this article as: B.T. Hopkins, E. Bame, N. Bell, T. Bohnert, J.K. Bowden-Verhoek, M. Bui, M.T.
Cancilla, P. Conlon, P. Cullen, D.A. Erlanson, J. Fan, T. Fuchs-Knotts, S. Hansen, S. Heumann, T.J. Jenkins, C.
Gua, Y. Yiu, Y. Liu, M. Lulla, D. Marcotte, I. Marx, B. McDowell, E. Mertsching, E. Negrou, M.J. Romanowski,
D. Scott, L. Silvian, W. Yang, M. Zhong, Utilizing Structure Based Drug Design and Metabolic Soft Spot
Identification to optimize the in vitro potency and in vivo Pharmacokinetic Properties Leading to the Discovery of
Novel Reversible Bruton’s Tyrosine Kinase Inhibitors, Bioorganic & Medicinal Chemistry (2021), doi: https://
doi.org/10.1016/j.bmc.2021.116275
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Utilizing Structure Based Design and Metabolic Soft Spot
Identification to Optimize the in vitro Potency and in vivo
Pharmacokinetic Properties Leading to the Discovery of
novel Reversible Bruton’s Tyrosine Kinase Inhibitors.
Leave this area blank for abstract info.
Brian T. Hopkinsa,  Eris Bame a, Noah Bellb, Tonika Bohnert a, Jon K. Bowden-Verhoeka, Minna Buib, Mark T.
Cancillab, Patrick Conlona, Patrick Cullena, Daniel A. Erlansonb, Junfa Fanb, Tarra Fuchs-Knottsb, Stig Hansenb,
Stacey Heumannb, Tracy J. Jenkinsa, Chungang Gua, Ying Yiua, YuTing Liua, Mukush Lullaa, Douglas Marcottea,
Isaac Marxa, Bob McDowellb, Elisabeth Mertschinga, Ella Negroua, Michael J. Romanowskib, Daniel Scotta, Laura
Silviana, Wenjin Yangb, Min Zhongb.
a Biogen Inc., 225 Binney Street, Cambridge, MA 02142, United States.
b Sunesis Pharmaceuticals, Inc. 395 Oyster Point Boulevard, South San Francisco, CA 94080, United States

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Utilizing Structure Based Drug Design and Metabolic Soft Spot
Identification to optimize the in vitro potency and in vivo Pharmacokinetic
Properties Leading to the Discovery of Novel Reversible Bruton’s Tyrosine
Kinase Inhibitors.
Brian T. Hopkinsa,  Eris Bame a, Noah Bellb, Tonika Bohnert a, Jon K. Bowden-Verhoeka, Minna Buib,
Mark T. Cancillab, Patrick Conlona, Patrick Cullena, Daniel A. Erlansonb, Junfa Fanb, Tarra Fuchs-Knottsb,
Stig Hansenb, Stacey Heumannb, Tracy J. Jenkinsa, Chuck Guaa, Ying Yiua, YuTing Liua, Mukush Lullaa,
Douglas Marcottea, Isaac Marxa, Bob McDowellb, Elisabeth Mertschinga, Ella Negroua, Michael J.
Romanowskib, Daniel Scotta, Laura Silviana, Wenjin Yangb, Min Zhongb.
a Biogen Inc., 225 Binney Street, Cambridge, MA 02142, United States.
b Sunesis Pharmaceuticals, Inc. 395 Oyster Point Boulevard, South San Francisco, CA 94080, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Bruton’s tyrosine kinase (BTK) is an essential node on the BCR signaling in B cells,
which are clinically validated to play a critical role in B-cell lymphomas and various
auto-immune diseases such as Multiple Sclerosis (MS), Pemphigus, and rheumatoid
arthritis (RA). Although non-selective irreversible BTK inhibitors have been approved
for oncology, due to the emergence of drug resistance in B-cell lymphoma associated
with covalent inhibitor, there an unmet medical need to identify reversible, selective,
potent BTK inhibitor as viable therapeutics for patients. Herein, we describe the
identification of Hits and subsequence optimization to improve the physicochemical
properties, potency and kinome selectivity leading to the discovery of a novel class of
BTK inhibitors. Utilizing Met ID and structure base design inhibitors were
synthesized with increased in vivo metabolic stability and oral exposure in rodents
suitable for advancing to lead optimization.
Available online
Keywords:
Bruton’s Tyrosine Kinase (BTK)
Fragment based screen, Computer aid drug design
(CADD), Met ID, Metabolic Soft Spot Switching
09 Elsevier Ltd. All rights reserved.
———
 Corresponding author. E-mail address: Brian.Hopkins@biogen.com

1. Introduction
To improve the drug-like properties, the central phenyl and urea
motifs were targeted for removal. The crystal structure of 2
indicated the urea moiety made a H-bond with Asp-539 while
orienting the substituted distal phenyl motif to extend into the
Bruton’s tyrosine kinase, a member of the Tec protein tyrosine
kinase (PTK)1 family, is critical for B cell development. More
than two decades ago, mutants in the BTK gene2-3 on the X
chromosome were found to be responsible for XLA patients’
inability to develop an adaptive immune response to opportunistic
infections. This genetic validation of BTK as a target for
modulating B cell development set the stage for identification of
small molecules BTK inhibitors as therapeutics for treatment of
auto-immune diseases such as rheumatoid arthritis (RA),4 multiple
sclerosis (MS),5-6 and B cell malignancies. 7
21
“H3” pocket. Utilizing in silico docking studies, a number of
structurally diverse molecules were designed which exhibited
binding poses enabling interactions with both the hinge region and
the Asp-539 residue. For example, replacing the di-aryl urea
moiety with the diarylamine motif was anticipated to preserve the
key H-bond interaction with Asp-539, although the docking
studies indicated the distal phenyl would be oriented away from
Tyr-551. Similarly, replacing the central phenyl moiety with a
flexible glycine linker was predicted to maintain the H-bond
interaction with Asp-539 while again positioning the distal phenyl
moiety away from the “H3” pocket.
In 2013 the FDA approved ibrutinib,8 a covalent BTK inhibitor for
the treatment of chronic lymphocytic leukemia (CLL) and mantle
cell lymphoma (MCL). Subsequently in 2017 and 2019
acalabrutinib9 and zanubrutinib10 were approved to treat CLL and
MCL as next generation irreversible BTK inhibitors exhibiting
superior clinical safety and tolerability due to their improved
kinome selectivity profiles. Although targeting BTK with covalent
inhibitors has proven successful, an unmet medical need has
emerged for new drugs which can target both the native and mutant
BTK protein (Cys-481 versus Ser-481), which has been
demonstrated to confer resistance11 associated with chronic dosing
of ibrutinib in patients. Thus, within the pharmaceutical industry
substantial resources have been deployed to identify reversible
drug candidates which do not require Cys-481 for activity, thereby
avoiding the potential for drug resistance due to site mutations in
the BTK kinase domain. To date six novel reversible BTK
candidates, fenebrutinib (GDC-0853),12 vecabrutinib (SNS-062),13
BIIB068,14 BMS-986142,15 ARQ531,16 AS-87117-18 and LOXO-
305,19 have advanced to the clinic for treatment of oncology and
auto-immune diseases such as systemic lupus erythematosus
(SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS).20
Route 1
H
N
H
N
NH2
Ph
Ph
a
b
N
N
N
N
Boc
Boc
N
5
4
3
NH
H
Route 2
N
Boc
N
Ph
HN
H
N
O
HN
Boc
c
d
N
N
H
N
N
N
N
6
Results and discussion
NH
8
7
NH
.
Previously we reported21 the utilization of a tethering fragment-
based screen which resulted in the identification of novel low
molecular weight hits which bound to BTK in a reversible fashion
as shown in Scheme 1. These hits were subsequently optimized to
improve both the in vitro potency and ADME properties leading
to the discovery of 2. This molecule was deprioritized based on an
unacceptably high predicted human dose.22 It was hypothesized
that optimizing the physicochemical properties, in particular
focusing on increasing the Fsp3 character,23 could increase the
solubility and in vitro cellular potency leading to improved drug-
like-properties and lowering the human predicted dose required for
efficacy to afford a potential candidate suitable for advancing into
development.
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, BINAP, t-BuONa, 24
hr, 80 °C, 68% yield; (b) i. HCl, 1,4-Dioxane, 24 hr, rt; ii. 4-Chloro-7H-
pyrrolo[2,3-d]pyrimidine, DIEA, DMF,100 °C, 6 hr, 83% yield; (c) 4-Chloro-
7H-pyrrolo[2,3-d]pyrimidine, DIEA, DMF,100 °C, 6 hr, 87% yield; (d) i.
HCl, 1,4-Dioxane, 24 hr, rt; ii. PhNHCH2CO2H, EDCI, HOBt, DIEA, DMF,
1 hr, rt, 81% yield.
Two synthetic routes were devised to enable the synthesis of both
targets in 3 steps starting from readily available starting materials
3 and 6. The bi-aryl amine 5 was synthesized utilizing a Buchwald-
Hartwig coupling24 of the aniline 3 with palladium acetate and
BINAP to afford the bi-aryl amine intermediate 4 in 68% yield.
The Boc group was removed under acidic conditions and the
product was reacted with 4-chloro-pyrrolopyrimidine in the
presence of excess DIEA to give 5 in 56% yield from 3. For the
synthesis of the glycine analog 8, coupling of the commercially
available tert-butyl piperidin-3-ylcarbamate 6 and 4-chloro-
pyrrolo-pyrimidine afforded intermediate 7, and removal of the
Boc group followed by amide coupling using EDCI/HOBt gave 8
in 71% yield for the 3 steps.
2. Hit to lead optimization
Table 1. BTK IC50 values for derivatives containing alkyl and aryl linkers.
HN
N
R
N
N
Figure 1. Summary of fragment-based screen to identify novel BTK hits.

aIC50 values were determined using non-phosphorylated BTK protein in a
FRET assay.
Compd
#
BTK
R
IC50 (µM)a
With a viable synthetic route in hand, it was feasible to determine
whether replacing the original bi-aryl urea core with either the bi-
aryl anime or glycine moieties would afford active molecules as
predicted by the in silico docking studies. Although the observed
affinity for 5 (BTK IC50 = 2.8 µM) was 3-fold weaker than 9 (BTK
IC50 = 0.8 µM), this finding was encouraging as it confirmed that
the key H-bond interaction with Asp-539 could be achieved using
other functional group beside the urea moiety 9. To further
improve the potency for the new lead 5, a series of ortho, meta and
para substituted trifluoromethyl anilines were synthesized as it
was hypothesized that the distal phenyl motif was binding in a
putative lipophilic region. Unfortunately, analogs 10, 11 and 12
did not show an improvement in biochemical potency compared
to 5.
O
4
9
0.8
2.8
1
3
N
N
H
2
1
H
4
3
5
N
2
H
10
>20
4.7
N
H
CF3
11
12
N
H
CF3
CF3
In a similar fashion a series of glycine analogs containing a
trifluoromethyl group at the ortho, meta and para position on the
distal phenyl were synthesized. To our delight the original glycine
analog 8 showed a 2-fold improvement in potency compared to the
urea lead 9. Furthermore, in contrast to the bi-aryl amine series,
introducing a lipophilic trifluoromethyl group25 at either the ortho
(13) or meta (14) position exhibited improved potency, although a
significant loss in activity was observed with the para substituted
trifluoromethyl analog (15) which was attributed to unfavorable
steric interactions within the active site. The stage was now set to
further optimize the potency and drug-like properties for this novel
glycine containing series.
>20
N
H
H
4
N
H
2
N
0.39
1
3
8
O
H
N
13
0.34
0.12
5.6
N
H
O
CF3
H
N
14
15
N
H
CF3
CF3
Table 2. Enzyme activity for analogs with various aryl substituents.
O
O
R4
H
N
R5
R3
R2
N
H
H
N
N
H
phenyl group with 5- or 6-membered heteroaryl moieties such as
pyridyl, pyridinyl, triazoles or cycloalkyl motifs all resulted in a
loss in affinity (BTK IC50 > 10 µM) compared to compound 8 (see
supplemental data). Thus, being unsuccessful in identifying a
suitable replacement for the aniline moiety, the focus switched to
mitigating the risk associated with this potential toxicophore.29-30
Since the preliminary SAR indicated that substituents at the meta
positions would be preferable, a series of commercially available
meta substituted anilines were selected to probe the SAR within
this region of the protein. Introducing polar substituents such as a
nitrile 17, or trifluoromethylsulfone 18, resulted in a > 5 fold loss
in potency, while lipophilic moieties such as fluoro32 or chloro
substituents exhibited comparable potency to 8. With the
O
R1
N
N
N
N
H
Compd
#
Regio-substitutions
BTK IC50
(µM)a
0.4
R1
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
R2
H
R3 R4
R5
8
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
14
16
17
18
19
20
21
22
23
24
25
26
27
28
CF3
C(CH3)3
CN
SO2CF3
OCF3
F
0.12
0.71
1.7
0.86
5.4
0.11
0.08
0.22
1.4
0.036
0.91
0.019
0.041
0.011
discovery of the meta chloro analog 21 exhibiting a BTK IC50
=
0.08 µM as a racemic mixture, the team focused on further
improving the potency by incorporating a second substituent at
either the 1, 3, 4 or 5 position on the phenyl moiety. Introducing
a lipophilic substituent such as a fluoro atom at the 2, 4 or 6
positions led to a loss in potency, while a modest 2-fold increase
in potency was observed with the 3-chloro-5-fluorophenyl analog
24. To capitalize on this result two analogs containing the 3,5-
dichloro and the 3-chloro-5-trifluoromethyl substituted aniline
were synthesized to afford 26 and 27 which exhibited similar
biochemical potency (BTK IC50 = 0.019 µM and BTK IC50 = 0.041
µM respectively) as observed with 24. To confirm the absolute
stereochemistry required for activity, both the R and S enantiomers
were synthesized to afford the eutomer 28 with good biochemical
potency (BTK IC50 = 0.011 µM) and >20 fold selectivity versus
other members of the Tec family (TEC IC50 = 0.13 µM, ITK IC50
= 0.23 µM, TXK IC50 = 2.9 µM and BMX IC50 = 0.94 µM).
Incorporating the 3,5-dichloroaniline moiety improved the
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
H
H
H
H
H
F
H
Cl
CF3
Cl
H
H
H
Cl
aIC50 values were determined using non-phosphorylated protein in a FRET
assay.
Compound 14 represented a new chemical class with improved
physicochemical properties and lipophilic ligand efficacy26-28 (14
LLE = 3.9 versus 9 LLE = 1.9). However, anilines have been
reported to exhibit a positive signal for genotoxicity in the AMES
assay, 29-31 so it was deemed necessary to devise a strategy for de-
risking the aniline moiety. Our initial attempts to replace the

biochemical potency, which translated to cellular activity (28,
inhibition of B cell proliferation IC50 = 1.2 µM). In addition, the
genotoxic risk was mitigated as the 3,5-dichloroaniline33-34 had
been reported to be negative for mutagenicity when tested in an
AMES assay.
BTK
IC50
B cell
RLM,
HLM
CL %QH
Compd
#
R
proliferation
(µM)a
IC50 (µM)b
c
N
N
28
0.01
1.2
>95, >95
N
N
N
H
29
30
0.01
0.13
0.5
7.0
>95, >95
>95, >95
N
N
H
N
O
N
N
H
N
31
32
33
0.067
1.0
3.8
NA
>10
>95, >95
>95, >95
NA
H2N
H2N
N
N
N
N
N
0.48
H2N
N
Fig. 2. Co-crystal structure of compound 21 bound in the BTK kinase
domain.
aIC50 values were determined using non-phosphorylated BTK protein in a
FRET assay. bIC50 for B-cell proliferation were measured. Peripheral blood
mononuclear cell (PBMC’s). cPredicted hepatic oxidative clearance was
measured in liver microsomal fraction, QH hepatic blood flow (portal vein
plus hepatic artery).
A 1.8 Å co-crystal structure (Fig 2.) of 21 bound to Btk showed
the pyrrolopyrimidine binding to the hinge region with the pyrrolo
ring oriented towards the Thr gatekeeper and facilitating key
hydrogen bond interactions with both Tyr-475 and Glu-475. The
piperidine ring occupied the ribose pocket and appeared to be
making hydrophobic interactions with the glycine-rich loop. As
the molecule extended further into the ATP pocket, the glycine
linker was ideally positioned to act as a dual hydrogen bond
donor/acceptor with both the catalytic Lys-430 and Asp-539 and
oriented the distal phenyl ring to bind in a putative hydrophobic
region away from Tyr-551. Further analysis revealed that the distal
aryl was making a face to edge π interaction with Phe 413 and the
Replacing the pyrrolopyrimidine hinge binder with the pyrazolo-
pyrimidine afforded 29 which exhibited similar biochemical
potency and a modest increase in cellular potency (B cell
proliferation IC50 = 1.2 vs 0.5 µM), but no improvement in the
microsomal stability was observed. Since the pyrrolopyrimidine
hinge binder was presumed to be metabolized via oxidation at the
C6 position, we synthesized 30 containing the 5,7-dihydro-6H-
pyrrolopyrimidin-6-one hinge binder. Unfortunately, this
modification led to a loss in biochemical potency and
demonstrated no improvement in the in vitro human liver
microsomal stability (HLM). Finally, a series of monocyclic
hetero-aryl analogs containing the H-bond donor were synthesized
and shown to be less potent in the biochemical assay (BTK IC50 =
0.067 µM (31), BTK IC50 = 1.0 µM (32), BTK IC50 = 0.48 µM
(33)) and did not exhibit any improvements in microsomal stability
when tested in the in vitro RLM or HLM assays.
3-chloro substituent was rotated toward the p-loop region.
With an understanding of the pharmacophore required for affinity,
the focus shifted towards utilizing the crystal structure to improve
the in vitro potency while optimizing the ADME properties. From
an ADME perspective, it was anticipated that the
pyrrolopyrimidine hinge binder would be a potential metabolic
soft spot for this series as it was reported in the literature to
undergo metabolic oxidation on the pyrrole ring.35 Thus, to assess
the liver microsomal stability, a series of analogs were synthesized
to explore alternative hinge binders which would maintain the key
H-bond donor/acceptor while attempting to reduce the electron
rich character of this motif.
Table 3. Enzyme activity for analogs with different hinge binding motifs.
Cl
H
N
N
Cl
H
O
N
R

H
N
HO
Cl
NH2
N
N
H
N
N
N
N
HO
O
N
a
b
N
+
N
N
N
O
[O]
N
O
Cl
H
N
H2N
H
N
Cl
O
H
Pathway C
HO
N
H
N
Cl
N
H
N
RLM: <5%
HLM: 6%
29g
35
36
H
34
Cl
29
RLM: 0%
HLM: 3%
Cl
H
N
H
Pathway B
H
N
N
N
N
[O]
NH2
O
N
HO
Cl
N
N
N
N-dearylation
37
[O]
N
Cl
Cl
Cl
O
H
N
Pathway A
Cl
OH
N
H
29e
N
29f
H
3%
6%
RLM:
HLM:
RLM: <5%
HLM: 7%
Scheme 2. Reagents and conditions: (a) Ar-X, CuI, Cs2CO3, DMF, 90 °C, 12
hr 71% yield; (b) EDCI, HOBt, DIEA, DMF, 1 hr, rt, 81% yield.
Cl
N
H
N
H
N
N
N
Since the Met ID studies highlighted pathway A as a major route
of clearance in both rat and human liver microsomes, we explored
the SAR on the central region of the molecule. Based on the
analysis of the BTK co-crystal structures it was envisioned that
introducing a substituent on the alpha carbon of the glycine linker
may block metabolism at this central linker region while affording
improvement in potency via introducing conformational
constraints into the flexible linker region. To evaluate this, a series
of chiral N-aryl-α-amino acids building blocks were synthesized
via Ullmann couplings, followed by treatment with the 3-amino-
piperidine moiety as described in Scheme 2 to afford a series of
analogs shown in Table 3.
N
N
N
N
N
N-dearylation
O
O
H
OH
N
Cl
N
H
N
H
OH
RLM: 11%
HLM: 19%
RLM: <5%
HLM: 31%
29b
29a
Cl
[O]
N-dearylation
H
N
H
N
H
N
N
N
N
N
N
N
N
N
N
N
N
Reduction
N
Table 3. Summary of in vitro biological potency and metabolic stability for
analogs with substituents on the glycine linker.
OH
O
OH
OH
OH
N
H
N
H
N
O
O
O
RLM: 85%
HLM: 13%
Cl
29c
29d
RLM: rat liver microsomes
HLM: human liver microsomes
R2
R1
H
N
N
H
Cl
Fig. 3. In vitro Met ID profile for compound 29 incubated with human and rat
liver microsomes. All % values are relative LCMS peak areas after 30 minutes
incubation of 29.
O
N
N
To establish an in vitro-in vivo correlation (IVIVC) for this series,
compounds 28 and 29 were dosed IV at 1 mg/kg in 40% NMP in
Sprague Dawley rats and exhibited high plasma clearance (CL
%QH = >100) consistent with the in vitro RLM data. In addition,
metabolite identification (Met ID) studies were completed to
elucidate potential metabolic soft spots attributed to the high in
vivo clearance. The in vitro studies highlighted two major
pathways for the oxidative metabolism for this series (Figure 3).
In both HLM and RLM the major route of metabolism was via
pathway A, involving oxidation of the glycine linker to afford the
hemiaminal 29a and subsequent de-alkylation and reduction to
afford 29b. In addition, oxidation of both the distal phenyl and the
hinge binder motifs was observed presumably via pathways B and
C respectively.
N
N
H
BTK
IC50
B cell
IC50
Cmpd
#
H, R, LM
%QH
R1
R2
LLE
c
(µM)a
(µM)b
28
37
H
H
H
0.01
1.2
1.3
>95
>95
3.9
3.5
Me
H
0.014
38
39
40
Me
Me
H
>10
>10
-
-
-
-
-
-
Me
Et
0.005
0.46
>95
3.4
41
42
H
H
0.011
0.005
0.4
0.8
>95
>95
3.4
3.0
OH
43
H
0.06
5.0
>95
3.5
O
44
H
1.5
Na
>95
2.8
aIC50 values were determined using non-phosphorylated BTK protein in a
FRET assay. bIC50 for B-cell proliferation were measured. Peripheral blood
mononuclear cell (PBMC’s). C Predicted hepatic oxidative clearance was
measured in human and rat liver microsomal (LM) fraction, QH hepatic blood
flow (portal vein plus hepatic artery).

Although introducing alpha substituents did not improve the in
vitro liver microsomal stability, hydrophobic and polar moieties
such as ethyl 40, cyclopropyl 41 or the iso-propyl 43 led to analogs
with improved cellular potency while maintaining acceptable
ligand efficiency (LLE). In addition to improving potency,
introducing the alpha cyclopropyl substituent 41 exhibited modest
off-target selectivity when profiled in the DiscoverX kinome panel
(42/410 with > 90% inhibition at 10 µM) and > 10 fold selectivity
over the Tec family members (TEC Kd = 0.051 µM, TXK Kd =
0.86 µM, ITK Kd = 0.021 µM, BMX Kd = 3.8 µM). To understand
how the binding to BTK was influencing the overall kinome
selectivity for this series, we decided to first identify the key
differences within the sequences across the Tec family members
which explained the selectivity profiled observed with this series
of compounds.
exhibited differences in the side-chain residues for Btk compared
to other Tec family members. For example, within the activation
loop (AVL region), residue 542 is a leucine in BTK while it is a
conserved methionine in TEC, TXK, BMX and ITK. And in the
hinge region (UPH) shown to be important for binding to BTK,
residue 474 is Thr for BTK and BMX versus a Phenylalanine for
TEC, TXK and ITK. Although ITK contains a bulky hydrophobic
Pheylalanine 474 gatekeeper compared to the polar conserved
Threonine gatekeeper in the other TEC family members, this
difference did not impact the overall selectivity for ITK versus
BTK. Based on the analysis of the structural data we postulated
that differences in the sequence homology within TEC family
members could be utilized as a strategy for improving selectivity.
Thus, a series of hybrid analogs were designed to interrogate the
SAR in both the AVL and the UPH region of the protein.
Table 5. Summary of the in vitro biological potency as determined in the
enzymatic and cellular assays and the selectivity against the TEC family
members.
R2
H
N
R1
N
H
O
N
N
N
X
N
H
Fig. 4 Illustration of compound 45 bound in the BTK active site.
BTK, TEC,
ITK, TXK,
BMX
Table. 4 Comparing residues within the BTK protein with other TEC family
members.
BTK
IC50
B Cell
IC50
Cmpd
#
X
R1
R2
LLE
(µM)a
(µM)b
Kinase
Domain Residue
Kd (µM)c
BTK TEC TXK BMX ITK
428
430b
432c
408
409
414
415
A
A
A
A
A
0.005, 0.05,
0.02, 0.9, 3.8
41
CH
Cl
Cl
F
0.012
0.4
3.1
B4
K
K
K
K
K
BAC
I
I
I
I
I
Id
G
G
Ld
0.003, 0.03,
0.02, 0.85,
3.5
L
G
G
V
L
G
G
V
Id
G
G
V
L
G
G
V
45
46
47
CH
CH
N
Cl
Cl
Cl
0.01
0.034
0.011
0.34
1.7
3.5
2.3
3.6
GRL
UPH
0.04, 0.04,
0.04, 1.6, 3.1
CF3
Cl
416
V
V
V
V
V
472
474a
475
476
I
I
T
E
Fd
I
T
E
Fd
I
L
Fd
E
Fd
0.006, -,
0.09, 2.5, 7.2
0.19
T
E
Y
T
E
Y
0.009, 0.05,
0.19, 5.7,
>10
48
49
N
N
Cl
Cl
F
0.011
0.057
0.2
4.2
2.4
477
M
M
M
Id
M
LWH
CTL
480
525
526
527
G
R
N
C
G
R
N
C
G
R
N
C
G
R
N
C
G
R
N
C
0.014, 0.07,
0.03, 3.6, 5.6
CF3
0.82
aIC50 values were determined using non-phosphorylated BTK protein in a
FRET assay. bIC50 for B-cell proliferation were measured with peripheral
blood mononuclear cells (PBMC’s). C KD were determined in DiscoverX®
KINOMEscanTM assays.
528
L
L
L
L
L
Introducing the pyrazolopyrimidine hinge binder led to an
improvement in the cellular potency and LLE while affording
modest selectivity versus other TEC family members such as ITK,
TXK and BMX, but unfortunately, the lead compounds 47 and 48
exhibited low metabolic stability in both the in vitro HLM and
RLM assays (CL %QH = > 90%).
539
D
L
D
D
D
D
AVL
542c
Md
Md
Md
Md
B4, Beta strand 4; BAC, Beta4-alphaC connector; GRL, glycine rich loop;
UPH, upper hinge; LWH, lower hinge; AVL, activation loop, CTL, Catalytic
loop. A Gatekeeper residue. B Catalytic Lysine. c Residues that comprise R-
spine. dDifferences in sequence from BTK sequence.
The co-crystallography structure revealed 20 residues within 3 Å
of compound 45, and alignment of the sequences for the Tec
family members identified two regions (UPH and AVL) which

F
Table 6. Summary of the in vitro biological potency and in vivo
clearance when dosed IV in a rat PK study.
H
N
N
Cl
F
H
O
OH
Cl
N
N
H
H
N
N
N
RLM: 7%
HLM: 36%
N
NH2
F
N
H
O
O
N
N
N
N
N
H
N
-dearylation
[O]
48
RLM: 55%
HLM: 19%
RLM: 7%
HLM: <5%
48a
N
N
N
N
N
-dearylation
48f
N
N
H
Cl
H
[O]
R
F
HO
H
N
H
[O]
N
OH
N
Cl
N
H
O
O
Dehydrogenation
N
N
N
N
RLM: 6%
HLM: <5%
N
N
RLM: 5%
HLM: 10%
N
N
N
H
N
N
N
N
F
H
H
BTK
IC50
B Cell Rat IV
48b
48e
Cmpd
#
R
IC50
CL
%QH
F
c
H
(µM)a (µM)b
H
N
N
HO
N
Cl
N
Cl
H
-2H
O
H
HO
O
N
N
N
RLM: 8%
HLM: 15%
RLM: <5%
HLM: 14%
N
N
N
N
N
N
H
N
N
48c
H
50
0.009
0.004
0.1
>90
>90
48d
N
H
O
O
RLM: rat liver microsomes, HLM: human liver microsomes
N
N
Fig. 5. In vitro Met ID profile for compound 48 incubated with human and rat
liver microsomes.
The lack of hydrolysis metabolism of carboxamide 48 in in vitro
in human and rat liver microsome and in rat hepatocytes suggested
that hydrolytic enzymes such as amidases were not involved in
metabolism of 48. The major pathways of metabolism identified
for compound 48 were via oxidation of the piperidine moiety
(48c), the cyclopropyl moiety (48e), and the distal aryl (48a, 48f),
in addition to N-dearylation at the glycine alpha carbon to afford
48b. It appeared that introducing the alpha substituent on the
glycine linker resulted in the switch of major metabolism to two
new sites, including the piperidine motif (48c and 48d) and
enhanced metabolism at the distal aryl (48a and 48f). To further
interrogate the potential for soft spot switching, a series of analogs
were designed to potentially block metabolism of the piperidine
and alpha-amino amide linker by introducing deactivating
substituents or conformational constraints within the molecule.
H
N
N
H
0.018
51
52
CF3
N
H
N
N
H
0.005
0.001
0.058
0.023
>90
>90
O
O
N
N
H
53
54
The BTK co-crystal structure suggested that introducing a methyl
substituent on the amide nitrogen or at the 4-position of the
piperidine ring may help improve the potency via interacting with
the p-loop while introducing steric constraints which could block
metabolism in this region of the molecule. Alkylating the amide
linker with a methyl group led to a modest improvement in cellular
potency but unfortunately 50 demonstrated high in vitro and in
vivo clearance (rat IV CL %QH = >90%). Substituting the
piperidine at the 4 position with either a methyl or the
trifluoromethyl moiety resulted in an improvement in biochemical
and cellular potency as predicted based on the co-crystal structure
but again both analogs exhibited high in vivo clearance (rat IV CL
%QH = >90%). We speculated that introducing conformational
rigidity via the formation of the 5,6 fused saturated ring system or
cyclizing the amide linker as a γ-lactam may improve the in vivo
clearance while maintaining the potency. Thus, replacing the
piperidine moiety with the novel 6,5-diazabicyclic moiety
afforded 53 which again was shown to exhibit good cellular
potency but poor in vitro and in vivo metabolic stability (rat IV CL
%QH = >90%). Fortunately, it was discovered that the γ-lactam 54,
which had reasonable biochemical potency (Btk IC50 = 0.015 µM),
also demonstrated moderate to low in vivo clearance (rat IV CL
%QH = 32, Table 6) when dosed IV at 0.25 mg/kg in 40% NMP
as a 1:1 mixture of diastereomers in a rat PK study.
N
N
N
H
0.015
1.14
48
O
N
aIC50 values were determined using non-phosphorylated BTK protein in a
FRET assay. bIC50 for B-cell proliferation were measured with peripheral
blood mononuclear cells (PBMC’s). c Predicted hepatic oxidative clearance
was measured in rat liver microsomal (LM) fraction, QH hepatic blood flow
(portal vein plus hepatic artery).

3. Lead optimization
NH2
a
b
N
O
N
N
Boc
Boc
55
56
F
d
c
N
N
I
N
Cl
H
O
N
O
N
Boc
Boc
57
58
F
Fig. 6 An overlay of the crystal structures of compounds 21 (blue/green) and
65 (brown/yellow) bound to BTK kinase.
F
N
N
Cl
H
e or g
O
h
N
N
With a viable synthesis in hand to enable further optimization of
the cyclic amide series, attention switched to establishing the SAR
which would confer good biochemical and cellular potency,
kinome selectivity, and reduced in vivo clearance. An overlay of
the co-crystal structures for 21 and 65 (racemic des-halo analog,
BTK IC50 = 0.19 µM) indicated that the new cyclic amide series
exhibited a binding pose where the alpha amino-lactam moiety
maintained the unique bi-dentate HBD/HBD interactions with the
catalytic Lys-430 and Asp-539 while orienting the distal phenyl
ring to bind in a putative hydrophobic pocket similar to the binding
pose observed with the glycine series (Figure 6). Based on the
structure, a series of analogs were synthesized as single
enantiomers designed to re-explore the distal phenyl moiety since
based on the prior SAR (table 4) this region of the molecule was
shown to be very amenable for modulating the biochemical
potency, kinome selectivity and physicochemical properties.
N
Cl
H
N
O
X
N
N
H
N
R
60 X = N, R
54 X = N, R
or
= CH2OCH2OCH2CH2SiMe3
= H
59
f
62 X = CH, R
= H
F
N
N
Cl
H
O
N
N
N
X
N
H
63 X = N
64 X = CH
Scheme 3. Reagents and conditions: (a) i. C5H8OBrCl, Et3N,
CH2Cl2, 0 °C, 3 hr 80% yield; ii. NaH, THF, 0 °C to 70 °C, 3 hr,
90% yield; (b) TMEDA, TMSCl, Ph-CH3, I2, 0 °C to rt, 18 hr, 60%
yield; (c) 3-Chloro-5-fluoroaniline, NaH, THF, 60 °C, 3 hr, 45%
yield; (d) TFA, CH2Cl2, rt, 2 hr, 91% yield; (e) i. C12H19ClN4O2Si,
n-butanol, Et3N, 80 °C, 2 hr, 85% yield; (f) i. HCl, 1,4-dioxane, 70
°C, 1 hr, 95% yield; (g) i. C4H4ClN3, n-butanol, Et3N, 80 °C, 2 hr,
79% yield; (h) Preparative chiral SFC separation.
Both the cyclic amide and glycine series exhibited similar SAR
trends with respect to modification on the distal aryl motif. For
example, compounds 63 and 66 containing the 3-fluoro-5-
chlorophenyl and the 3,5-dichlorophenyl moieties demonstrated
biochemical potencies within < 2 fold of 42 and 43, although the
biochemical potency observed within the lactam resulted in a
significant loss in cellular potency (PBMC CD69 IC50 = 1.2 µM
(63)) compared to the acyclic series (PBMC CD69 IC50 = 0.09 µM
(47)). This result was attributed to the increase in the lipophilicity
(63 Log D = 4.6 versus 47 Log D = 4) resulting in an increase in
plasma protein binding (% Fu = 1 (63) versus % Fu = 6 (47)) and
a decrease in the kinetic solubility at pH 6.8 (<1 µg/mL versus 7
µg/mL respectively). Since the lead compound 54 had been dosed
in vivo as a mixture of diastereomers, it was necessary to first
determine if the single enantiomer would exhibit similar in vivo
stability compared to the diastereomeric mixture. Unfortunately,
when 63 was dosed IV in a rat PK study, the eutomer demonstrated
higher in vivo plasma clearance (63, rat IV CL %QH = 70)
compared to 54 (rat IV CL %QH = 48 as a 1:1 mixture of
diastereomers), thus reiterating the need to complete the PK
studies using chiral material. In addition, switching the distal
phenyl to the 3,5-dichlorophenyl moiety afford 66 with high
clearance (rat IV CL %QH = >100) when dosed as an IV cassette
(0.25 mg/kg, 40% NMP) in Sprague Dawley rats. The addition of
a fluoro atom at the para position of the distal phenyl 67 and 68
improved the in vivo clearance compared to 63 and 65. This
finding was consistent with the Met ID studies as the 4 position
had been identified as a potential site for metabolism (Schemes 3
and 5) but unfortunately, these analogs were less potent in the
The synthesis of chiral cyclic amide 63 began with acylation of
tert-butyl piperidin-3-ylcarbamate 55 upon treatment with 5-
bromovaleryl chloride, which was subsequently treated with NaH
in DMF at rt for 24 hr to facilitate cyclization to 56 in a 72% yield.
Amination at the α-position of the γ-lactam was accomplished in a
two-step sequence, beginning with treatment of 56 with TMEDA,
TMSCl and iodine to afford intermediate 57 which was
subsequently reacted with 3-chloro-5-fluoroaniline under basic
conditions to afford 58 as a 1:1 mixture of diastereomers in an
overall 27% yield. Deprotection of the Boc group with HCl
followed by N-arylation upon treatment of the SEM protected 4-
chloro-pyrazolopyrimidine under mild basic conditions, followed
by deprotection afforded 61 which was separated using SFC to
afford the eutomer 63.

biochemical assay Finally switching the hinge binder from the
pyrazolo-pyrimidine to the pyrrolo-pyrimidine afforded 64 with
moderate in vivo clearance (CL %QH
= 50), adequate
Table 8: Summary of biological, ADMR and physical chemical properties for
compound 64.
bioavailability (% F = 48) when dosed at 5 mg/kg as an amorphous
solid as suspension formulation in carboxymethyl cellulose
(CMC)/Tween, good biochemical potency (BTK IC50 = 0.004 µM)
and cellular activity (PBMC CD69 IC50 = 0.63 µM).
F
Table 7. Summary of the in vitro biological potency and in vivo
clearance when single enantiomers are dosed IV in a rat PK
study.
N
N
Cl
H
O
N
N
N
R
N
N
H
N
64
O
H
N
N
Assay
Compd 64
N
X
N
H
Physical-chemical properties: MW, Log D,
442, 4.5, 77, 4.3
0.004
PSA, LLE26
Btk
IC50
B cell
IC50
Rat
IV CL LLE
Cmpd
#
Biochemical potency: BTK IC50 µMa
R
X
(µM)a (µM)b %QH
c
Cellular potency: B-cell proliferation anti-
F
0.63
IgM+IL-4 IC50 µMb
63
66
67
68
N
0.010
0.008
0.053
0.018
1.95
1.62
4.85
3.39
70
>90
28
3.8
3.9
3.5
3.3
Cl
Selectivity against TEC family members:
0.007, 0.013,
0.083, 1.6, 2.7
Cl
BTK, ITK, TEC, TXK, BMX KD µMc
N
N
N
Cl
Aqueous kinetic solubility: µg/mL at pH 6.8
1, >40
28, 1.4
3.6, 18
and 1.5d
F
F
Permeability: Caco-2 A-B Papp (10-6
cm/sec), efflux ratioe
Cl
Cl
Metabolic stability: Human, rat in vitro liver
F
f
microsomal CL %QH
18
Cl
Plasma protein binding: %Fu human, ratg
1.1, 1.9
F
64
CH
0.004
0.630
53
4.3
IV (dose 1 mg/kg), CL (ml/min/kg),
29, 55, 1.6, 0.8
632, 0.5, 48%
Cl
CL %QH Vss (L/kg), T1/2 (hr)
Rath
PK
aIC50 values were determined using non-phosphorylated BTK protein in a
FRET assay. bIC50 for B-cell proliferation were measured with peripheral blood
mononuclear cells (PBMC’s). Predicted hepatic oxidative clearance was
PO (dose 5 mg/kg suspension),
AUCinfi (hr*mg/mL, Tmax (hr), F (%)
c
measured in rat liver microsomal (LM) fraction, QH hepatic blood flow (portal
vein plus hepatic artery).
aIC50 values were determined using non-phosphorylated protein in
a
biochemical assay. bIC50 for B-cell proliferation were measured with peripheral
blood mononuclear cells (PBMC’s). CKd were determined in DiscoverX®
KINOMEscan™ assays. dKinetic solubility was measured using Analiza®
miniaturized shake flask assay. eTransepithelial transport across Caco-2/TC-7
monolayers. FPredicted hepatic oxidative clearance was measured in liver
microsomal fraction and corrected for microsomal binding (%QH hepatic blood
In addition to the favorable biological and in vivo pharmacokinetic
properties, compound 64 inhibited 18/459 kinases by >80% when
tested at 0.7 µM (100-fold over the BTK Kd) and modest BTK
selectivity versus other members of the Tec family members (Tec
Kd = 0.013 µM, ITK Kd = 0.082 µM, TXK Kd = 1.6 µM, BMX Kd
= 2.7 µM) when tested at DiscoverX. Profiling of the ADME
properties indicated that 64 exhibited weak CYP inhibition toward
3A4 (CYP IC50 = 1.3 µM) only when tested using testosterone, and
no activity against other isoforms (IC50 = >10 µM). Compound 64
showed good intrinsic permeability with no efflux and pH
dependent solubility (kinetic pH 3 and 6.8 >50 ug/mL & <1
ug/mL). Applying a minimal PBPK model, the human PK
properties for 64 were simulated with a predicted CL of 1.4
mL/min/Kg (from liver microsomal stability), Vss (from
physicochemical properties) of 1.28 L/Kg, and an estimated ka of
0.9/hr-1 (from observed ka in preclinical species for this series).
g
flow (portal vein plus hepatic artery). Plasma protein binding (PPB) 50 at a
concentration of 2 µM was assessed for rat, and human plasma, via rapid
h
equilibrium dialysis (RED). PK studies were conducted in Sprague Dawley
rats dosing IV as a solution in 40% NMP and PO as a suspension in
carboxymethyl cellulose (CMC)/Tween.
Conclusion
Herein, we describe the use of structure-based design to identify
BTK inhibitors which were optimized focusing on improving the
physical chemical properties to increase potency, kinome
selectivity, and in vivo plasma stability leading to the discovery of
a novel cyclic lactam series. The most advanced molecule from the

hit-to-lead campaign was compound 64, which exhibited adequate
biological and ADME properties to transition into lead
optimization, ultimately leading to the discovery of vecabrutinib,
which was taken into clinical to evaluate as a treatment for patients
diagnosed with relapsed/refractory chronic lymphocytic leukemia
(CLL).
27.44, 27.86, 28.41, 53.70, 79.98, 98.53, 98.78, 104.96, 105.21,
109.09, 135.44, 149.37, 154.71, 154.76, 162.54, 164.98, 169.95;
LCMS (ESI): m/z 370.1 (M+H)+ -tertButyl C17H21N3ClFO3:
370.1.
. (3'R)-3-((3-chloro-5-fluorophenyl)amino)-[1,3'-bipiperidin]-
2-one (59). To solution of 58 (1.8 g, 4.2 mmol) in CH2Cl2 (20
mL) was added TFA (3 ml) and was stirred at rt 2 hr. The
reaction solution was adjusted to pH = 7 - 8 with NaHCO3,
extracted with EtOAc (50 mL X 3), dried (Na2SO4), filtered and
concentrated in vacuo to afford 59 as colorless oil (1.5 g, 91%).
1H NMR (500 MHz, CD3OD-d4) δ 6.49 (s, 1H), 6.35 (d, J=1.22
Hz, 1H), 6.32-6.34 (m, 2H), 4.31-4.41 (m, 1H), 4.01 (dd, J=6.64,
1.07 Hz 1H), 3.35-3.42 (m, 1H), 3.27-3.34 (m, 1H), 2.92 (br t,
J=11.60 Hz 2H), 2.68 (dt, J=4.58, 11.75 Hz, 1H), 2.40-2.50 (m,
1H), 2.17-2.27 (m, 1H), 1.86-1.93 (m, 5H), 1.70-1.76 (m, 2H),
1.54-1.69 (m, 2H). 13C NMR (126 MHz, CD3OD-d4) δ 172.4,
166.3, 164.4, 152.2, 136.4, 110.1, 104.6, 99.2, 54.7, 53.5, 46.4,
43.2, 28.0, 27.4, 22.0; LCMS (ESI): m/z 326.2 (M+H)+.
General. All reagents and solvents were purchased from
commercial sources and were used without further purification.
Reaction progress was monitored by LCMS. Flash
chromatography was performed with Isco Combiflash Companion
system using pre-packed silica gel columns. 1HNMR spectra were
recorded at 400 MHz and 600 MHz using a Bruker AVANCE III
spectrometer with a BBFO probe. Compound purities were
estimated by reversed-phase C18 HPLC, and SFC with a UV
detector at 214 and 254 nm, and the major peak area of each tested
compound was ≥95% of the combined total peak area.
tert-Butyl (R)-2-oxo-[1,3'-bipiperidine]-1'-carboxylate (56). To
a solution of tert-butyl (R)-3-aminopiperidine-1-carboxylate (20.0
g, 100 mmol) and Et3N (20.2 g, 200 mmol) in CH2Cl2 (200 mL)
was added 5-bromopentanoyl chloride (23.8 g, 120 mmol) at 0oC.
The mixture was stirred at rt for 3 hr and concentrated in vacuo,
diluted with water and extracted with EtOAc (200 mL X 3). The
combined organic layers were dried (Na2SO4), filtered and
concentrated in vacuo to afford an oil (25.0 g, 68.5 mmol) which
was dissolved in THF (200 ml) and treated with NaH (4.9 g, 205
mmol) at 0 oC. The mixture was stirred at 70 oC for 3 hr under N2,
quenched with water, extracted with EtOAc (200 mL X 3), the
organic layer were dried (Na2SO4), filtered and concentrated in
vacuo to afford a residue which was purified by silica gel column
(Petroleum ether: EtOAc = 1:9 (v/v)) to afford tert-butyl (R)-2-
oxo-[1,3'-bipiperidine]-1'-carboxylate 57 (18.0 g, 90%) as yellow
oil. 1H NMR (400 MHz, CDCl3) δ 3.25 (m, 1H) 3.18 (m, 2H), 2.82
(t, J = 11.8, 11.8 Hz, 1H), 2.81 (m, 1H), 2.57 (br s, 1H), 2.41 (br
s, 2H), 1.77 (br s, 4H), 1.64 (m, 6H), 1.45 (s, 9H); 13C NMR (400
MHz, CDCl3) δ 20.74, 23.37, 27.32, 28.42, 32.63, 43.28, 79.80,
(3'R)-3-((3-Chloro-5-fluorophenyl)amino)-1'-(1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-
4-yl)-[1,3'-bipiperidin]-2-one (60). A solution of 59 (1.5 g, 4.6
mmol),
4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
pyrazolo[3,4-d]pyrimidine (1.3 g, 4.6 mmol), Et3N (0.9 g, 9
o
mmol) in n-butanol (20 mL) was heated to 80 C for 2 hr, the
mixture was allowed to cool to rt diluted with water and extracted
with EtOAc (20 mL X 3), the combined organic layers were dried
(Na2SO4), filtered and concentrated in vacuo to afford a residue
which was purified silica gel column (Petroleum ether: EtOAc =
1: 9 (v/v)) to give (3'R)-3-((3-Chloro-5-fluorophenyl)amino)-1'-
(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-
d]pyrimidin-4-yl)-[1,3'-bipiperidin]-2-one 69 (1.8 g, yield: 85%)
as white solid..1H NMR (500 MHz, CD3OD-d4) δ 8.27 (s, 1H),
8.26 (d, J=1.22 Hz, 1H), 6.49 (s, 1H), 6.28-6.37 (m, 2H), 5.67 (s,
2H), 4.86 (s, 2H), 4.63-4.81 (m, 1H), 4.31-4.46 (m, 1H), 4.06
(ddd, J = 6.41, 10.53, 12.36 Hz, 1H), 3.63 (t, J = 8.24 Hz, 2H),
3.39-3.53 (m, 2H), 3.32-3.37 (m, 1H), 3.10 (br s, 1H), 2.18-2.31
(m, 1H), 1.86-2.06 (m, 5H), 1.62-1.78 (m, 2H), 0.83-0.92 (m, 2H),
-0.07 (d, J = 1.83 Hz, 9H); 13C NMR (126 MHz, CD3OD-d4) δ
172.7, 166.3, 164.4, 158.3, 156.5, 156.1, 152.2, 136.4, 135.7,
110.0, 104.6, 101.9, 99.2, 76.2, 68.1, 54.8, 52.9, 52.7, 43.7, 28.5,
28.0, 25.8, 22.1, 18.7, -1.3; LCMS (ESI): m/z 574.1 (M+H)+;
C27H37N7ClFO2Si: 574.1.
154.83, 169.72; ESI-MS (m/z): 227.3, calcd for [M+ H]+
-
tertButyl C11H18N2O3: 227.3.
tert-Butyl
(3'R)-3-((3-chloro-5-fluorophenyl)amino)-2-oxo-
[1,3'-bipiperidine]-1'-carboxylateboxylate (58). To a solution
of 56 (4.5 g, 16 mmol) in PhCH3 (80 mL) was added
N1,N1,N2,N2-tetramethylethane-1,2-diamine (8.7 g , 97.5 mmol)
and TMSCl (7.0 g, 65 mmol) at 0 oC and allowed to stir for 6 hr,
followed by the addition of I2 (8.0 g, 32.5 mmol) and stirred at rt
overnight under N2. The mixture was quenched with water,
extracted with EtOAc (100 mL X 3) and the combined organic
layers were dried (Na2SO4), filtered and concentrated in vacuo to
(3'R)-3-((3-Chloro-5-fluorophenyl)amino)-1'-(1H-
pyrazolo[3,4-d]pyrimidin-4-yl)-[1,3'-bipiperidin]-2-one (54).
To a solution of 60 (1.8 g, 3.1 mmol) in MeOH (15 mL) was added
4 N HCl in 1,4-dioxane (1 mL) and stirred at 70 oC for 1 hr. The
reaction extracted with EtOAc (50 mL X 3) and the combined
organic layers were dried (Na2SO4), filtered, concentrated in
vacuo to afford an oil which was purified silica gel column
(Petroleum ether: EtOAc = 1: 9 (v/v)) to give (3'R)-3-((3-Chloro-
5-fluorophenyl)amino)-1'-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)-
give
tert-butyl
(3'R)-3-iodo-2-oxo-[1,3'-bipiperidine]-1'-
carboxylate 57 (4.0 g, 60 %) as yellow solid. Intermediate 57 (4.0
g, 10 mmol) was dissolved in THF (20 mL) and added slowly at
rt to a solution of 3-chloro-5-fluoroaniline (2.2 g, 15 mmol) and
NaH (0.36 g, 15 mmol) in THF (40 mL). The mixture was heated
at 60 oC for 3 hr under N2, quenched with NH4Cl, extracted with
EtOAc (100 mL X 3), dried (Na2SO4), filtered and concentrated in
vacuo to afford a residue which was purified by silica gel column
(Petroleum ether: EtOAc = 1:1 (v/v)) to give tert-Butyl (3'R)-3-
iodo-2-oxo-[1,3'-bipiperidine]-1'-carboxylate 58 as yellow oil (1.8
g, 45%). 1H NMR (400 MHz, CDCl3) δ 6.34 (br d, 8.2 Hz, , 1H),
6.39 (br s, 1H), 6.22 (dt, J = 11.1, 2.0, 2,0 Hz, 1H), 5.23 (s, 1H),
3.78 (br m, 1H), 3.32 (ddd, J = 10.2, 9.8, 4.9 Hz, 2H), 2.88 – 2.76
(m, 1H), 2.47 (br s, 1H), 1.98 – 1.86 (m, 2H), 1.83 (br m, 1H),
1.72 (br d, J = 11.8 Hz, 1H), 1.65 (br m, 1H), 1.60 (s, 3H), 1.46
(br d, 9H); 13C NMR (400 MHz, CDCl3) δ 20.70, 21.07, 26.44,
1
[1,3'-bipiperidin]-2-one 61 as white solid (1.0 g, yield: 95%). H
NMR (500 MHz, CD3OD-d4) δ 8.21 (d, J = 3.66 Hz, 2H), 6.45-
6.54 (m, 1H), 6.28-6.39 (m, 2H), 4.62-4.82 (m, 2H), 4.31-4.43 (m,
1H), 4.06 (ddd, J = 6.10, 10.07, 13.12 Hz, 1H), 3.46 (dtd, J = 6.10,
12.17, 17.78 Hz, 2H), 3.35 (br s, 1H), 3.10 (br s, 1H), 2.19-2.29
(m, 1H), 1.88-2.04 (m, 5H), 1.62-1.78 (m, 2H); 13C NMR (126
MHz, CD3OD-d4) δ 172.7, 166.4, 164.4, 158.4, 156.5, 156.3,
152.2, 136.4, 135.5, 110.0, 104.6, 101.2, 99.2, 54.8, 52.9, 43.8,
28.6, 28.4, 28.0, 25.8, 22.1. LCMS (ESI): m/z 444.1 (M+H)+;
C21H23ClFN7O: 444.1. Anal. Calcd for C21H23N7O1Cl1F1 0.5H2O:
C, 55.69; H, 5.34; N, 21.65; Found C, 55.65; H, 5.19; N, 21.30.

(3R,3'R)-3-((3-chloro-5-fluorophenyl)amino)-1'-(7H-
104 in 100 µL of complete RPMI medium per well) were cultured
with serially diluted BTK inhibitors as indicated in 96-well round
bottom plates (Corning, Corning, NY) for 30 min at 37 °C.
Following incubation of B cells with BTK inhibitors, cells were
stimulated for 3 days with F(ab’)2 goat anti-human IgM antibody
(10 µg/mL; Jackson ImmunoResearch, West Grove, PA) and
recombinant human IL-4 (X µg/mL; R&D Systems, City, State).
At the end of day 3, an equal volume of CellTiter-Glo® Reagent
(Promega, Madison, WI) was added to the cell culture and the
contents were mixed on an orbital shaker for 2 min at room
temperature. The samples were then rested for an additional 10
min at RT and the emitted luminescent signals were measured with
a SpectraMax luminometer (Molecular Devices, Sunnyvale, CA).
The luminescence relative units (RLU) from the inhibitor-treated
samples were then graphed versus log10 inhibitor concentration in
GraphPad Prism and the IC50 value was determined.
pyrrolo[2,3-d]pyrimidin-4-yl)-[1,3'-bipiperidin]-2-one (64).
To a solution of 59 (0.23 g, 6.9 mmol) and 4-chloro-7H-
pyrrolo[2,3-d]pyrimidine (0.1 g, 6.9 mmol) in DMF (3.45 mL)
was added Hunigs base (0.27 g, 20.7 mmol, 0.36 mL) and stirred
at 100 °C for 6 hr. The reaction was diluted with water and
extracted with EtOAc (50 mL X 2) and the combined organic
layers were dried (Na2SO4), filtered, concentrated in vacuo to
afford an oil which was purified silica gel column (Petroleum
ether: EtOAc = 1: 9 (v/v)) to give (3'R)-3-((3-Chloro-5-
fluorophenyl)amino)-1'-(1H-pyrrolo[3,4-d]pyrimidin-4-yl)-[1,3'-
bipiperidin]-2-one 61 as an off white solid (0.24 g, yield: 79%).
The diastereomers were separated via SFC chiral chromatography
(CHIRALPAK IB 30x250mm, 5um Method: 40% MeOH w/
0.1% DEA in CO2 (flow rate: 100mL/min, ABPR 120bar, MBPR
40psi, column temp 40 deg C) to afford (3R)-3-(3-chloro-5-
fluoro-anilino)-1-[(3R)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3-
piperidyl]piperidin-2-one (96.60 mg, 218.10 umol, 31.58% yield)
(Peak 1) and (3S)-3-(3-chloro-5-fluoro-anilino)-1-[(3R)-1-(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)-3-piperidyl]piperidin-2-one
(0.0706 g, 159.40 umol, 23.08% yield) (Peak 2) as off-white
solids. 1H NMR (500 MHz, CD3OD-d4) δ 8.12 (s, 1H), 7.08 (d, J
= 3.66 Hz, 1H), 6.61 (d, J = 3.66 Hz, 1H), 6.49 (s, 1H), 6.34 (dd,
J = 2.14, 3.36 Hz, 1H), 6.32 (d, J = 1.83 Hz, 1H), 4.61-4.72 (m,
2H), 4.36-4.46 (m, 1H), 4.02 (dd, J = 6.10, 9.77 Hz, 1H), 3.33-
3.48 (m, 2H), 3.18 (t, J = 11.90 Hz, 1H), 2.95-3.04 (m, 1H), 2.16-
2.25 (m, 1H), 1.83-1.98 (m, 5H), 1.62-1.74 (m, 2H). 13C NMR
(126 MHz, CD3OD-d4) δ 172.6, 166.3, 164.4, 158.4, 152.5, 152.2,
151.7, 136.4, 122.6, 110.1, 104.7, 104.5, 102.7, 99.2, 54.7, 52.9,
47.4, 43.5, 28.8, 28.0, 26.1, 22.0. LCMS (ESI): m/z 444.1
(M+H)+; C22H24ClFN6O: 443.1. Anal. Calcd for C22H24N6O1Cl1F1:
C, 59.66; H, 5.46; N, 18.97; Found C, 59.18; H, 5.31; N, 18.70.
Methods for Caco-2 permeability assay: Caco-2 cell
monolayers were grown to confluence on microporous
polyethylene membranes in 24-well BD Falcon™ insert plates.
The permeability assay buffer was HBSS containing 25 mM
HEPES at pH of 7.4. The buffer in the receiver chamber also
contained 1% bovine serum albumin. DMSO stock solutions were
prepared at 5 μM in assay buffer from 10 mM stock solution, and
added to the apical side and basolateral side. Cells were incubated
at 37 ºC with 5% CO2 in a humidified incubator. Samples were
collected from the donor chambers at 0 and 120 minutes, and
receiver chambers at 120 minutes. After samples were collected,
100 µM lucifer yellow was added to all apical chambers, and fresh
buffer was added to the basolateral chambers. The permeability of
lucifer yellow was measured for each monolayer for 60 minutes to
ensure the integrity of the cell monolayers during the test
compound flux period. The apparent permeability coefficient, Papp
,
was calculated as follows:
Papp = (dCr /dt) x Vr / (A x CE)
Methods for BTK biochemical activity assay: Compound
inhibition was measured after monitoring the amount of
phosphorylation of a fluorescent-labeled polyGAT peptide in the
presence of active BTK enzyme (Millipore 14-552), ATP, and an
inhibitor. The BTK kinase reaction was performed a black 96-well
plate (Corning 3694). For a typical assay, a 24 µL aliquot of a
ATP/peptide master mix (final concentration; ATP 10 µM,
polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10
mM MgCl2, 200 µM Na3PO4, 5 mM DTT, 0.01% Triton X-100,
and 0.2 mg/mL casein) was added to each well. Next, 1 µL of a 4-
fold, 40X compound titration in 100% DMSO solvent was added,
followed by 15 µL of BTK enzyme mix in 1X kinase buffer (with
a final concentration of 0.25 nM). The assay was incubated for 30
minutes then stopped with 28 µL of a 50 mM EDTA solution.
Aliquots (5 µL) of the kinase reaction were transferred to a low
volume, white 384-well plate (Corning 3674), and 5 µL of a 2X
detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20
antibody, Invitrogen PV3552) was added. The plate was covered
and incubated for 45 minutes at room temperature. Time resolved
fluorescence (TRF) on Molecular Devices M5 plate reader (332
nm excitation; 488 nm emission; 518 nm fluorescein emission)
was measured. IC50 values were calculated using a four-parameter
curve fit with 100% enzyme activity determined from the DMSO
control and 0% activity from the EDTA control.
Where dCr /dt is the permeation rate of cumulative concentration
in the receiver compartment versus time in µM s-1, Vr is the
volume of the receiver compartment in cm3, A is the area of the
insert (0.31 cm2 for 24-well insert), CE is the calculated
experimental concentration (Time = 0) of the dosing solution in
µM, determined via LC/MSMS.
Acknowledgments
The team is grateful for Biogen Inc. and Sunesis
Pharmaceuticals, Inc. for providing financial support to this work.
Appendix A. Supplementary Material
Supplementary data to this article can be found online at
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Declaration of interests
☒ 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.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: