Optimization of novel reversible Bruton's tyrosine kinase inhibitors identified using Tethering-fragment-based screens

Article abstract of DOI:10.1016/j.bmc.2019.05.021

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for

Full text of DOI:10.1016/j.bmc.2019.05.021

Accepted Manuscript
Optimization of Novel Reversible Bruton’s Tyrosine Kinase Inhibitors Identi-
fied Using Tethering-Fragment-Based Screens
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, Douglas Marcotte, Bob McDowell, Elisabeth Mertsching, Ella
Negrou, Kevin L. Otipoby, Urjana Poreci, Michael J. Romanowski, Daniel
Scott, Laura Silvian, Wenjin Yang, Min Zhong
PII:
S0968-0896(19)30374-8
https://doi.org/10.1016/j.bmc.2019.05.021
BMC 14908
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 March 2019
9 May 2019
13 May 2019
Please cite this article as: Hopkins, B.T., Bame, E., Bell, N., Bohnert, T., Bowden-Verhoek, J.K., Bui, M., Cancilla,
M.T., Conlon, P., Cullen, P., Erlanson, D.A., Fan, J., Fuchs-Knotts, T., Hansen, S., Heumann, S., Jenkins, T.J.,
Marcotte, D., McDowell, B., Mertsching, E., Negrou, E., Otipoby, K.L., Poreci, U., Romanowski, M.J., Scott, D.,
Silvian, L., Yang, W., Zhong, M., Optimization of Novel Reversible Bruton’s Tyrosine Kinase Inhibitors Identified
Using Tethering-Fragment-Based Screens, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.bmc.2019.05.021
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Optimization of Novel Reversible Bruton Tyrosine Kinase
Inhibitors Identified using Tethering-Fragment-Based
Screens
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, Douglas Marcottea, Bob McDowellb, Elisabeth Mertschinga, Ella Negroua, Kevin L. Otipobya, Urjana Porecia,
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
Fragment Based Screening
and Library Synthesis
OH
F
O
O
R'
H
H
S
n
R
N
N
N
N
Hit
Hit-to-lead and
lead optimization
N
N
S
HN
N
N
H
5
identification
H
H
O
N
N
Y
S
R
N
N
S
N
N
R'
X
H
n
H
5
N
BTK IC50 = 11 M
N
H
50
=
BTK IC50 0.001 M
n
X
Y
= 1, 2; = N; = -C(O)-, -
S(O2)-, -CH2C(O)-, -NHC(O)NH-

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Optimization of Novel Reversible Bruton’s Tyrosine Kinase Inhibitors Identified
Using Tethering-Fragment-Based Screens
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, Douglas Marcottea, Bob McDowellb, Elisabeth
Mertschinga, Ella Negroua, Kevin L. Otipobya, Urjana Porecia, 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
Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous
clinical trials have been reported using covalent inhibitors to target Bruton’s tyrosine kinase
(BTK) for oncology indications. However, a formidable challenge for the pharmaceutical
industry has been the identification of reversible, selective, potent molecules for inhibition of
BTK. Herein, we report application of Tethering-fragment-based screens to identify low
molecular weight fragments which were further optimized to improve on-target potency and
ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors
suitable for pre-clinical proof-of-concept studies.
Available online
Keywords:
Bruton’s Tyrosine Kinase (BTK)
Fragment screen, Computer aid drug design (CADD)
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. E-mail address: Brian.Hopkins@biogen.com

BTK inhibitors fenebrutinib (GDC-0853),27 vecabrutinib (SNS-
062),28 BMS-986142,29 ARQ-531,30 and AS-871,31 with varying
degree of kinome selectivity. Even though the next-generation of
covalent inhibitors have demonstrated improved kinome
selectivity compared to ibrutinib, other potential risks such as
immune-mediated drug hypersensitivity reactions32 (IDHR’s) or
emergence of drug resistance33 associated with chronic dosing led
to our strategic decision to develop reversible BTK inhibitors for
treatment of immunological indications. To further maximize the
probability of identifying novel chemical matter, a fragment-
based screening34 approach was chosen which was anticipated to
yield low molecular-weight fragments more amenable for
optimization of potency, kinome selectivity, and drugability.35
Herein we report the use of a Tethering-fragment-based screen to
identify low molecular weight building blocks as hits which were
optimized using traditional medicinal chemistry approaches and
computer aided drug design (CADD) leading to the discovery of
compounds such as 50, which exhibit biological potency, kinome
selectivity, and in vivo rat PK properties applicable for proof of
concept (POC) in rodent efficacy studies.
1. Introduction
The origin of Bruton’s Tyrosine Kinase (BTK)1 in the scientific
literature can be traced back to Dr Ogden Bruton’s report in 1952
of
a
rare and potentially fatal disorder called
agammaglobulinemia. Patients with this inherited disease have
low to absent B cells and therefore lack the ability to generate an
adaptive
immune
response
to
produce
protective
immunoglobulins. These patients suffer from opportunistic
bacterial infections and recurrent sinopulmonary infections. The
cause of this immunodeficiency, now known as Bruton's
syndrome or X-linked agammaglobulinemia (XLA), was not
fully elucidated for another forty years, when it was discovered
that XLA patients’ inability to generate an adaptive immune
response and protective antibodies was due to mutations in the
BTK gene2 on the X chromosome.
Humans and mice3 with genetic lesions in BTK show defects in
early B-cell development and mature B-cell activation due to
defective signaling from the B-cell antigen receptor (BCR).4
BTK connects the immunoreceptor tyrosine-based activation
motif (ITAM)-containing BCR signaling complex with
downstream signaling events including phospholipase Cγ
activation, the Ca2+ response, and NF-kB activation.5 In myeloid
cells such as monocytes and neutrophils, BTK has been
demonstrated to be important for Fcγ receptors signaling
(FcγRs).6 Both B cells and myeloid cells are thought to have
important functions in various autoimmune diseases such as
rheumatoid arthritis7 (RA),7 multiple sclerosis (MS),8 and
systemic lupus erythematosus (SLE).9 For example, Rituxan®,
which binds the B-cell-restricted surface marker CD20 and
induces antibody-mediated depletion of B cells, is therapeutically
effective in RA and MS patients.10 Furthermore, neutrophils are
thought to be aberrantly activated by autoantibody-containing
immune complexes in SLE patients.11 Targeting BTK signaling
should therefore inhibit B-cell activation by the BCR and
myeloid-cell activation by FcγR and may provide therapeutic
benefit for patients with autoimmune diseases.12
2. Results and discussion
Figure 1. General workflow for the fragment screen.
BTK is a member of the tyrosine kinase expressed in
hepatocellular carcinoma (Tec) family of kinases consisting of
five members: (1) Tec,13 (2) BTK, (3) interleukin-2 (IL-2)-
inducible T-cell kinase (ITK;14 also designated Emt15 or TSK),
(4) resting lymphocyte kinase (Rlk;16 also known as TXK17), and
(5) bone-marrow tyrosine kinase gene on chromosome X
(BMX;18 also referred to as ETK). Tec kinases are primarily
expressed in hematopoietic cells,19 although expression of BMX
and Tec has been detected in epithelial cells,20 endothelial
cells,18a and fibroblasts. The Tec family members share a high
degree of sequence homology in the Src Homology (SH)3 and
SH2 and N-terminal domains21 which presents a formidable
challenge when designing small molecules that selectively target
BTK but not the other family members. The first generation of
BTK inhibitors in the clinic were irreversible inhibitors
containing an acrylamide Michael acceptor that covalently
modifies the BTK protein at Cys 481 and led to the development
of ibrutinib,22 and acalabrutinib,23 approved by the US FDA in
2012 and 2017 for the treatment of Chronic Lymphocytic
Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Ibrutinib
has been demonstrated to covalently bind to 9 out of > 400
kinases across the entire kinome (i.e., BTK, BMX, B lymphocyte
kinase (BLK), epidermal growth factor receptor (EGRF), human
epidermal growth factor receptor 2 (HER2), HER4, ITK, Janus
kinase 3 (JAK3), and TEC, and subsequent next generation
molecules have been developed to improve overall kinome
selectivity.24 This effort has led to the discovery of a number of
clinical candidates such evobrutinib (M2951) and spebrutinib25
(AVL-292) which are selective irreversible inhibitors, a pseudo-
irreversible inhibitor PRN-1008,26 and more recently, reversible
Sunesis’ Tethering fragment based lead discovery plathform,36
was used to identify low molecular weight monophores
(fragments) that bound in the ATP binding site (Figure 1).
Initially, the protein was treated with various custom synthesized
low molecular weight “Extenders” functionalized with an
acrylamide to react with native Cys 481 residue as well as a
disulfide moiety.37 The assay conditions utilized in the screen
enabled monophores exhibiting weak affinity for the active site
to covalently cross-link with the protein complex via a disulfide
bond
with
the
extender,
and
the
resulting
protein/Extender/monophore complex was analyzed by mass
spectrometry. Upon deconvolution of the hits, the resulting
monophores (fragments) which demonstrated affinity to the
target, along with analogs and additional simple fragments, were
reconfigured into small libraries of molecules constructed using a
variety of alkyl linkers (Table 1 and Scheme 1). Compound 5
exhibited reversible binding to BTK with a biochemical IC50 = 11
µM.

Cl
N
Cl
N
O
a
b
N
N
N
H
R
N
N
Ts
H
1
2
N
N
N
n = 4
NH2
O
H
n = 4
HN
HN
N
N
N
H
H
N
c
N
N
N
N
Ts
Ts
3
4
H
H
N
N
HN
N
d
O
N
5
N
H
Scheme 1. Reagents and conditions: (a) C7H7SO2Cl, NaOH, H2O,
(CH3)2CO, rt, 10 h, 95% yield. (b) i. tert-butyl 6-aminohexylcarbamate,
DIEA, EtOH, 80 οC, 6 h; ii. HCl, EtOH, rt, 3 h, 81% yield. (c) PhNCO, base,
CH2Cl2, rt, 8 h, 90%. (d) K2CO3, THF/H2O (4:1 (v/v)), 80 οC, 5 h, 78% yield.
Once novel, low molecular weight fragments were identified
using Tethering, the next task was to understand how these
fragments bound in the BTK active site. It was hypothesized that
the hits were binding in a “DFG-in” conformation. This was
supported by in silico docking studies (performed using Glide
with the default option)38 which indicated that the
pyrrolopyrimidine moiety was oriented towards the hinge region
whereas the distal hydrophobic urea extended into the “H3”
pocket (Figure 2) similarly to the binding mode published for the
BTK inhibitors (CGI-1746) from CGI/Genentech.39
Figure 2. View of compound 5 docked in the active site of BTK and
superimposed with CGI-1746 highlighted in tan (PDBID:3OCS).
3. Hit to lead optimization
Table 1. Enzyme activity for derivatives containing alkyl and aryl linkers.

Thus, the hybrid compound 11 was synthesized in a similar
fashion as shown in Scheme 1 except the tert-butyl (6-
aminohexyl)carbamate tether was replaced with commercially
available (3-piperidin-3-yl-phenyl)-carbamic acid tert-butyl ester
to give the racemic mixture which had a BTK IC50 = 0.8 µM.
Separation of the isomers by chiral chromatography afforded the
eutomer 13 with a BTK IC50 = 0.4 µM whereas the enantiomer
12 was inactive (BTK IC50 > 20 µM). Successful replacement of
the flexible acyclic linker with the aryl-piperidine scaffold
yielded the lead compound 13 that was amenable for further
optimization.
BTK
Compd
R
IC50 (µM)a
H
N
5
6
7
11
16
19
N
H
N
N
H
H
HN
CO2Et
+
CO2Et
b
NH
Br
H
N
H
N
a
8
12
B
N
O
O
N
H
N
H
N
16
14
15
9
8.3
2.0
H
H
CO2Et
H
N
N
N
10
Cbz
N
N
Cbz
N
c
d
H
N
11
12
13
0.8
>20
0.4
N
N
N
Boc
Boc
17
18
19
N
H
N
N
R
N
Ts
H
N
H
N
H
H
N
N
H
N
S
O
N
O
N
e
f
N
aIC50 values were determined using non-phosphorylated BTK protein in
FRET assay.
N
N
N
N
20
11
N
The original hit identified from the screen contained a disulfide
linker which was deemed unsuitable for further development, so
a series of 40 analogs were synthesized replacing the disulfide
linker with substituted aliphatic acyclic, cyclic, and aromatic
motifs. Analogs containing the hexyl 5, pentyl 6, or hex-3-yne 7
linkers (Table 1, entries 1-3) had similar biochemical potency
NH
Ts
Scheme 2. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DME, 80 οC,
12 h, 80% yield; (b) i. H2, AcOH, 10% Pd/C, 150 psi, 100 οC, 87% yield
(crude); ii. (Boc)2O, NaHCO3, H2O, THF, rt, 12 h, 70% yield. (c) i. LiOH,
H2O, THF, 0 οC to rt 6 h, 90% yield (crude); ii. Benzyl alcohol, DPPA, DIEA,
PhCH3, 80 οC, 8 h, 81% yield; (d) i. HCl, 1,4-dioxane, MeOH, rt, 4 h; ii. 4-
Chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine, DIEA, DMF, 100 οC, 75%
yield; (e) i. H2, 10% Pd/C, MeOH, rt, 15 psi; ii. Phenyl isocyanate, DIEA,
DMF, rt, 1 h; (f) K2CO3, THF/H2O (4:1 (v/v) 80 οC, 5 h, 71% yield.
(BTK IC50 = 11 µM (5), BTK IC50 = 16 µM (6), and BTK IC50
=
19 µM (7)), consistent with the docking studies and the
hypothesis that the tethered fragments were binding in two
distinct regions of the protein approximately 6 to 7 Å apart. To
further probe this hypothesis, several analogs were synthesized
containing a variety of di-substituted aryl moieties designed to
introduce conformational constraint into the central region of the
molecule. The meta substituted phenyl moiety 8 was preferred as
compared with other regio-isomers, and simply inverting the
connectivity of the linker to the 3-(2-aminoethyl)aniline (9) gave
a similar potency (BTK IC50 = 12 µM vs BTK IC50 = 8.3 µM). A
further 4-fold improvement in activity was achieved when the
aminoethylaniline linker 9 was replaced with the 3-(piperidin-3-
yl)propan-1-amine 10 (BTK IC50 = 2 µM). Based on in silico
docking it was hypothesized that combining both the substituted
aryl and piperidine motifs could increase potency since the
overlays of both 9 and 10 indicated the piperidine and phenyl
moieties were binding in different regions of the ATP pocket.
Having identified the lead molecule 13 with sub-micromolar
biochemical potency, the next task was to determine whether
further improvements in potency could be achieved. To
accomplish this goal, a synthetic route was developed to facilitate
rapid exploration of the SAR from a common intermediate. The
pyridyl-biaryl 16 (Scheme 2) was synthesized via a Suzuki
coupling using commercially available ethyl 3-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)benzoate
(14)
and
3-
bromopyridine (15).40 Subsequent hydrogenation41 and treatment
with Boc anhydride gave the corresponding piperidine 17,
followed by saponification. The resulting acid moiety was
converted to the Cbz-protected aniline 20 via a Yamata
modification of the Curtius rearrangement and the piperidine was
selectively deprotected to generate the secondary amine which
was treated with 4-chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine to

afford intermediate 19. The Cbz group was removed by
palladium-mediated hydrogenation to afford the aniline which
gave the phenyl urea upon treatment with phenyl isocynate.
Finally, removal of the tosyl protecting group under mildly basic
conditions gave the desired compound 11 in 19% overall yield.
To facilitate further exploration of SAR at both hinge and distal
regions of the ATP binding pocket, the orthogonally protected
compound 18 was used as a key intermediate.
R4
R5
R3
R2
O
N
N
H
H
R1
N
N
N
Table 2. Enzyme activity for the aniline substituted BTK inhibitors.
N
H
R
N
Compd
Regio-substitutions
R2 R3 R4 R5 R6
BTK IC50
(uM)
H
N
N
11
27
28
29
30
31
32
33
34
35
36
37
H
N
H
H
F
H
H
Cl
H
H
H
H
N
H
H
F
H
H
Cl
H
H
H
H
H
H
N
H
H
F
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.8
2.7
3.2
>10
0.5
1.2
1.3
0.12
1.8
2.3
N
H
N
BTK
Compd
11
R
IC50 (µM)a
CF3
t-Butyl
0.46
0.14
O
Ph
0.8
aIC50 values were determined using non-phosphorylated protein in a FRET
assay.
N
H
O
21
22
23
4.9
19
45
Retaining the N,N’-diaryl urea linker, the emphasis switched to
optimizing alternative regions of the molecule such as the distal
aryl. Initial attempts to replace the aryl group with a pyridine
moiety such as 27, 28, or 29 (Table 3) led to a loss in potency
regardless of the position of the nitrogen atom to the urea linker.
Introducing fluorine atoms at the ortho, meta, and para positions
on the distal aryl all maintained comparable potency to 11.
Furthermore, it was observed that ortho substituted chloro analog
33 afforded greater than 10-fold more potency relative to meta or
para chloro-substituted analogs 34 and 35. Although, the
improvement in potency correlated with an increase in the
lipophilicity, there were other factors contributing to the SAR.
For example introducing the ortho triflouromethyl group 36 led
to a 3 fold decrease in potency, which was attributed to the
electron withdrawing effect of trifluoromethyl group since the
ortho substituted t-butyl analog 37 exhibited similar activity to
33. In addition, it was speculated that ortho substituents such as
the chloro and tert-butyl groups 37 and 33 were restricting
rotation about the Ar-N bond in the phenylurea, thus the gain in
potency may be attributed to the distal moiety adopting a
favorable conformation necessary for binding to the active site.
Ph
O
S
O
Ph
O
Ph
HN
24
4.1
N
O
O
25
26
44
68
Ph
N
H
CN
N
Ph
N
H
aIC50 values were determined using non-phosphorylated protein in a FRET
assay.
With a viable synthetic route in hand, the focus was turned
toward identifying suitable replacements for the urea linker.
Based on in silico docking studies, the N,N’-diaryl urea moiety
was hypothesized to form hydrogen bonds with the conserved
Lys 430 and Asp 539 (Figure 1), which in turn facilitated the
distal aromatic moiety to bind in the Tyr 511 pocket. A series of
analogs were synthesized replacing the N,N’-diaryl urea with
other linkers such as an amide 21 or a sulfonamide group 22; but
all these structural modifications led to diminished potency
Table 4. Enzyme activity for ortho substituted aryl urea BTK inhibitors.
(Table 2). In addition, a series of
urea biosisoters43
(benzimidazole 24, squaric acid 25, or N-cyanoguanidine 26)
were evaluated but also resulted in decreased potency relative to
11.
Table 3. Enzyme activity for SAR of BTK inhibitors with distal aryl
modifications.

R1
which needed to be further optimized in addition to improving
the cellular activity.
.
O
N
N
H
H
R
N
N
4. Lead identification
N
N
H
NO2
NO2
a
NO2
TBDMS
Stereo-
chemistry
BTK
Compd
37
R
R1
O
IC50 (µM)a
b
N
HN
HN
R/S
enantiomers
N
N
N
t-Butyl
H
0.14
>10
0.2
NH
N
N
N
N
R/S
enantiomers
38
39
40
41
H
H
H
H
N
Ts
CN
Ts
46
44
45
R/S
enantiomers
N
H
H
NH2
N
N
R/S
enantiomers
0.1
O
O
O
TBDMS
TBDMS
d
F
c
N
N
R/S
enantiomers
0.02
N
N
N
N
N
N
N
N
N
R/S
enantiomers
47
48
42
H
0.55
N
N
Ts
Ts
R
N
43
H
0.009
H
H
N
enantiomer
N
aIC50 values were determined using non-phosphorylated protein in a FRET
assay.
HO
O
N
F
e
N
To further explore the nature of the interaction preferred for
activity analog 38 containing 2-methylpropanitrile, a known tert-
butyl isostere43 was synthesized (Table 4). This resulted in a loss
of activity, although other lipophilic replacements such as
cyclopropyl 39 or cyclopentyl 40 moieties were as potent as 37.
Substituting the ortho cyclopentyl 40 for the pyrrolidine 41
increased the electron density on the distal aromatic ring giving a
further 7-fold improvement in potency as a racemic mixture.
However, enlarging the 5-membered ring to a six membered
piperidine moiety 42 resulted in a loss in potency most likely due
to unfavorable steric interactions with the protein. The racemic
mixture 41 was separated by chiral chromatography to afford the
eutomer 43 with a BTK IC50 = 0.009 µM, while the enantiomer
(not shown) was > 166-fold less potent. Compound 43 inhibited
51/468 kinases by > 90% when tested at 10 µM (1000-fold over
the BTK IC50) using the DiscoverRX kinome panel. Furthermore,
43 had good selectivity for BTK versus other members of the Tec
family (ITK KD = 0.4 µM, TEC, TXK, and BMX, KD = > 1 µM)
which is consistent with the compound binding to the BTK “H3”
pocket. Having demonstrated the ability to optimize the
biochemical potency while maintaining modest kinome
selectivity, 43 was further profiled as a potential candidate for in
vivo studies. The in vitro ADME profiling of 43 identified a
number of potential concerns such as poor solubility (< 1 µg/mL
at pH 6.8), CYP450 inhibition (3A4 IC50 = 1 µM, 2C9 IC50 = 1.8
µM, 2C19 IC50 = 2.2 µM, 1A2 IC50 = 2.2 µM), and high in vitro
N
N
49
NH
Scheme 3. Reagents and conditions: (a) 4-Chloro-7-tosyl-7H-pyrrolo[2,3-
d]pyrimidine, DIEA, DMF, 80 οC,
h, 52% yield; (b)
(CH3)3CSi(CH3)2OCH2CHO, NaBH3CN, MeOH, AcOH, 100 ºC, 3 h, 78%
yield; (c) H2 Raney Ni, rt, 0.5 h, 96% yield; (d) i. Triphosgene, CH2Cl2,
NaHCO3, rt, 1h; ii. 2-Fluoro-6-(pyrrolidin-1-yl)aniline, DIEA, CH2Cl2, 30%
4
ο
yield; (e) i. 12 N HCl, MeOH, 2h rt; ii. K2CO3, water, MeOH, 65 C, 2 h,
68% yield.
The low solubility and poor metabolic stability of 43 led the team
to synthesize compounds containing a substituted piperazine
motif, designed to modulate the physicochemical properties
while maintaining the key pharmacophores required for binding.
The piperazine moiety was anticipated to decrease the in vitro
clearance by introducing polarity45 in the central region of the
molecule while helping to further improve the solubility. The
synthesis of these compounds began with commercially available
2-(3-nitrophenyl)piperazine 44, which was treated with tosyl
protected 4-chloro-pyrrolopyridine under mild basic condition to
afford the desired isomer 45 in a 52% overall yield. The amine
was protected and the nitro group was reduced to aniline 47 using
Raney Ni and subsequently treated with triphosgene and 2-
fluoro-6-(pyrrolidin-1-yl)aniline to give 48. The TBDSM group
clearance44 (CL %Qh(corrected
human 67, rat 56)
for protein binding)

was removed with HCl followed, by Tosyl deprotection under
mild basic conditions to afford 49 as a racemic mixture.
bioavailability, based on calculated hepatic extraction ratio from
in vivo observed IV plasma clearance and assumption of Fa x FG
= 1, was approximately 89%. Unfortunately, this compound
exhibited low oral exposure (AUCinf 1889 hr*ng/mL),
corresponding to only 12% oral bioavailability (%F) when dosed
Table 5: Summary of the biological, ADME and physical chemical
properties for compound 50.
as an amorphous solid in
a
carboxymethyl cellulose
F
(CMC)/Tween suspension formulation, suggesting poor fraction
of absorption. The poor oral absorption of 50 most likely is due
to a combination of poor permeability and moderate efflux
(Table 5) poor solubility/dissolution, and high intestinal
metabolism (compounds in series were metabolized primarily by
CYP3A4, making them susceptible to high intestinal
metabolism).
O
HO
N
N
N
N
H
H
N
N
N
H
N
Assay
Compd 50
Physical-chemical properties
MW, Log D, PSA, LLE43
544, 2.6, 113, 5.7
0.001
BTK biochemical IC50 (µM)a
B-cell proliferation
0.24
anti-IgM+IL-4 IC50 (µM)b
Off-target kinases
0.005, 0.42, 0.53, 0.09
ITK, TEC, TXK, BMX KD (µM)c
Kinetic solubility
Figure 3: Simulated predicted human concentration-time profiles (Simcyp
Simulator v 15).
8, 94
4, 11
(µg/mL at pH 6.8 and 1.5)d
Caco-2 A-B Papp (10-6 cm/sec),
efflux ratioe
Prediction of human PK was performed using physicochemical
parameters, observed permeability, and in vitro liver microsomal
CLint, with the Simcyp simulator (version 15). Simulator47
predicted a 250 mg BID (dark red) would achieve target
engagement level of 50% (light green) for 12 hr in humans.
Minimal PBPK model was applied to generate simulated human
PK with predicted CL of 4.8 mL/min/kg (from in vitro liver
microsomal stability), Vss (from physicochemical properties and
method 1) of 0.5 L/kg, and ka of 1 hr-1 (from observed ka in
preclinical species for this series) and Fa = 0.01 (from observed
Human, rat liver microsomal
53, 10
f
stability CL %QH
PPB %Fu human, ratg
1.0, 0.13
aIC50 values were determined using non-phosphorylated protein in
a
b
biochemical assay. IC50 for B-cell proliferation were measured. Peripheral
blood mononuclear cells (PBMC’s). CKD were determined in DiscoverRx®
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 flow (portal vein plus hepatic artery). gPlasma protein binding (PPB) 50
at a concentration of 2 µM was assessed for rat, and human plasma, via rapid
equilibrium dialysis (RED).
Fa in animals for this series). CL = in vivo clearance; Vss
Volume of distribution, ka = Absorption rate constant, Fa =
Fraction absorbed.
=
While compound 50 was predicted to have a low CL in humans
(~25% QH), due to poor Fa, the efficacious dose to maintain 50%
target engagement (EC50), as observed in the B-cell proliferation
assay was predicted to be high (250 mg BID; 500 mg daily)
(Figure 3).
The racemic mixture 49 was separated by chiral chromatography
to afford the eutomer 50, which exhibited biochemical (BTK IC50
= 0.001 µM) and cellular activity (PBMC CD69 IC50 = 0.24 µM)
while maintaining modest selectivity toward the Tec family
members and improved kinome selectivity, inhibiting only
21/468 kinases > 90% at 1 µM (i.e., screening concentration
1000-fold over the BTK IC50). In addition, the piperazine moiety
improved some of the in vitro ADME properties such as aqueous
kinetic solubility (8 µg/mL at pH 6.8), CYP450 inhibition (all
isoforms IC50 > 5 µM) and in vitro metabolic stability (Cl
%QH(corrected for protein binding) Human 53, Rat 10).
To evaluate the in vivo PK properties of the molecule, 50 was
dosed IV at 1 mg/kg in 40% NMP in Sprague Dawley rats.
Compound 50 demonstrated low plasma clearance (CL 6.2
mL/min/kg, % QH = 11), which correlated with the predicted rat
in vitro liver microsomal stability, corrected for protein binding.
The volume of distribution was observed to be low (Vss = 0.21
L/Kg), with a terminal half-life (t1/2) of 2.5 h. Estimated
Crystal structure.
A co-crystal of compound 50 bound to BTK was obtained at 1.7
Å resolution (Figure 4). The structure revealed the following
observations: (1) the pyrrolo-pyrimidine moiety acts as a dual
hydrogen bond donor/acceptor with residues Glu 475 and Tyr
476 in the hinge region, (2) the piperazine moiety binds in the
ribose pocket and orients the central phenyl ring toward the
glycine rich loop (p-loop), (3) the urea moiety forms a hydrogen
bond interaction with the Asp 539, and (4) the distal phenyl
moiety is twisted out of conjugation with the urea functional
group, allowing for the substituted aryl to bind into a putative
hydrophobic pocket which in turns facilitates the pyrrolidine ring
at the 2 position to extend into the “H3” Tyr 551 pocket. These

data confirmed that 50 bound to the ATP pocket of BTK in a
“DFG-in” confirmation.
2H), 2.39 (s, 3H). 13C NMR (400 MHz, CDCl3) δ: 156.9, 152.9,
152.2, 148.5, 145.5, 143.5, 135.0, 133.3, 129.7, 129.6, 128.3,
123.1, 122.1, 122.0, 105.0, 104.6, 59.5, 52.6, 46.2, 45.8, 21.7;
HR-ESI-MS (m/z): 479.1497, calcd for [M+ H]+ C23H22N6O4S:
479.1496.
4-(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-(3-
nitrophenyl)piperazin-1-yl)-7-tosyl-7H-pyrrolo[2,3-
d]pyrimidine
(46).
To
a
solution
of
4-(3-(3-
nitrophenyl)piperazin-1-yl)-7-tosyl-7H-pyrrolo[2,3-d] pyrimidine
45 (0.91 g, 1.9 mmol, 1.0 eq) in MeOH/AcOH (15:1, 16 mL) was
added 2-((tert-butyldimethylsilyl)oxy)acetaldehyde (0.65 g, 3.76
mmol, 2.0 eq) at rt. NaBH3CN (0.35 g, 5.64 mmol, 3.0 eq) was
added into the mixture. The mixture was stirred at 100 oC for 3 h,
allowed to cool to rt and adjusted pH = 8 with 3 N NaOH and
extracted with EtOAc (100 mL). The organic phase was washed
with brine, separated dried (Na2SO4), filtrated, and concentrated
in vacuo to afford a residue which was purified by silica gel
column (Petroleum ether/EtOAc = 3/1 (v/v)) to give 4-(4-(2-
((tert-butyldimethylsilyl)oxy)ethyl)-3-(3-nitrophenyl)piperazin-
1-yl)-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine 46 (0.94 g, 78%) as
Figure 4. Co-crystal structure of compound 50 bound in the BTK kinase
domain.
1
light yellow solid. H NMR (400 MHz, CDCl3) δ: 8.44 (s, 1H),
8.34 (s, 1H), 8.21 (dd, J = 8.0, 1.2 Hz, 1H), 8.09 (d, J = 8.0 Hz,
2H), 7.81 (d, J = 7.6 Hz, 1H), 7.59-7.53 (m, 2H), 7.32 (d, J = 8.0
Hz, 2H), 6.55 (d, J = 4.4 Hz, 1H), 4.62-4.58 (m, 2H), 3.69-3.64
(m, 2H), 3.58-3.55 (m, 1H), 3.47-3.40 (m, 1H), 3.33-3.30 (m,
1H), 3.09-3.03 (m, 1H), 2.61-2.51 (m, 2H), 2.42 (s, 3H), 2.27-
2.21 (m, 1H), 0.86 (s, 9H), 0.01 (s, 3H), 0.00 (s, 3H). 13C NMR
(400 MHz, CDCl3) δ: 156.5, 152.9, 152.1, 148.6, 145.4, 143.1,
135.1, 134.2, 129.8, 129.7, 128.3, 123.0, 122.9, 122.0, 104.9,
104.5, 66.0, 61.1, 56.5, 52.8, 52.4, 25.9, 21.7, 18.3, -5.3, -5.4;
HR-ESI-MS (m/z): 637.2628, calcd for [M+ H]+
C31H41N6O5SSi: 637.2623.
Conclusion
A fragment-based approach facilitated the identification of BTK
hits that were further optimized using both traditional medicinal
chemistry and structure-based design, resulting in a novel series
of reversible BTK inhibitors with good biochemical potency on-
target cellular activity for inhibiting B cell proliferation.
Although the lead compound 50 had acceptable cellular potency,
in vitro ADME properties, and in vivo PK in rat for preclinical
proof-of-concept studies this compound was deprioritized due to
high predicted dose in humans, necessary to maintain continuous
coverage of 50% target engagement (EC50) value. However, with
the discontinuation of the series, efforts were refocused on
optimization of the novel leads identified from the hit-to-lead
campaign, which subsequently led to the discovery and
development of clinical candidate vecabrutinib (SNS-062).28
3-(1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(7-tosyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-2-yl)aniline (47).
A
mixture of 4-(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-(3-
nitrophenyl)piperazin-1-yl)-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine
46 (0.92 g, 1.42 mmol, 1.0 eq) and Raney Ni® in THF (20 mL)
was stirred at rt under H2 for 0.5 h. The suspension was filtrated
and concentrated in vacuo to afford a residue 47 (0.82 g, 96%)
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.
1
which was used in the next step without further purification. H
NMR (400 MHz, CDCl3) δ: 8.26 (s, 1H), 7.97 (d, J = 8.4 Hz,
2H), 8.37 (d, J = 4.0 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.04 (t, J
= 8.0 Hz, 1H), 6.69 (d, J = 7.6 Hz, 1H), 6.65 (s, 1H), 6.53 (dd, J
= 7.6, 1.6 Hz, 1H), 6.44 (d, J = 4.0 Hz, 1H), 4.69 (d, J = 12.8 Hz,
1H), 4.51 (d, J = 12.8 Hz, 1H), 3.66-3.55 (m, 4H), 3.21-3.13 (m,
4H), 2.74-2.66 (m, 1H), 2.55-2.47 (m, 1H), 2.40 (s, 3H), 2.25-
2.19 (m, 1H), 1.75 (s, 9H), -0.10 (s, 3H), -0.11 (s, 3H). 13C NMR
(400 MHz, CDCl3) δ: 156.5, 153.0, 152.1, 146.8, 145.4, 141.7,
135.1, 129.7, 128.3, 121.6, 118.2, 114.7, 114.3, 104.9, 104.8,
67.1, 61.5, 56.3, 53.4, 53.0, 45.8, 25.9, 21.7, 18.3, -5.3, -5.4; HR-
ESI-MS (m/z): 607.2881, calcd for [M+ H]+ C31H43N6O3SSi:
607.2881.
4-(3-(3-Nitrophenyl)piperazin-1-yl)-7-tosyl-7H-
pyrrolo[2,3-d]pyrimidine (45).
A
mixture of 2-(3-
nitrophenyl)piperazine 44 (0.9 g, 4.35 mmol, 1.0 eq), 4-chloro-7-
tosyl-7H-pyrrolo[2,3-d]pyrimidine (1.4 g, 4.56 mmol, 1.1 eq) and
DIEA (0.84 g, 6.53 mmol, 1.5 eq) in DMF (15 mL) was stirred at
1-(3-(1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(7-tosyl-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-2-yl)phenyl)-3-(2-
fluoro-6-(pyrrolidin-1-yl)phenyl)urea (48). To a solution of 3-
(1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(7-tosyl-7H-
pyrrolo[2,3-d] pyrimidin-4-yl)piperazin-2-yl)aniline 47 (0.82 g,
1.32 mmol, 1.0 eq) in CH2Cl2 (20 mL) was added a saturated
solution of NaHCO3 (1 N, 10 mL) at rt, followed by the addition
of triphosgene (1.20 g, 4.0 mmol, 3.0 eq). The reaction mixture
was stirred at rt for 1 h. The organic phase was separated and
dried (Na2SO4). After filtration, 2-fluoro-6-(pyrrolidin-1-
yl)aniline (0.72 g, 4.0 mmol, 3.0 eq) and DIEA (0.81 g, 6.6
mmol, 5.0 eq) were added to the solution. The mixture was
refluxed for 2 h and then concentrated in vacuo to afford a
o
60 C for 4 h. The mixture was diluted with water (50 mL) and
extracted with EtOAc (100 mL). The organic phase was washed
with brine, dried (Na2SO4), filtrated and concentrated in vacuo to
afford a residue which purified by silica gel column (Petroleum
ether/EtOAc = 1/1) to afford 4-(3-(3-nitrophenyl)piperazin-1-yl)-
7-tosyl-7H-pyrrolo[2,3-d]pyrimidine 45 as light yellow solid (1.2
1
g, 52%). H NMR (400 MHz, CDCl3) δ: 8.43 (s, 1H), 8.38 (s,
1H), 8.18 (dd, J = 8.4, 1.2 Hz, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.80
(s, 1H), 7.57-7.50 (m, 2H), 7.29 (d, J = 8.0 Hz, 2H), 6.56 (d, J =
4.4 Hz, 1H), 4.71 (d, J = 12.4 Hz, 1H), 4.57 (d, J = 12.4 Hz, 1H),
3.98 (dd, J = 10.4, 2.8 Hz, 1H), 3.37-3.23 (m, 2H), 3.08-2.99 (m,

residue that was purified by silica gel column (petroleum
ether/EtOAc 2/1 (v/v)) to give 1-(3-(1-(2-((tert-
butyldimethylsilyl)oxy)ethyl)-4-(7-tosyl-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)piperazin-2-yl) phenyl)-3- (2-fluoro-6-
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.
=
(pyrrolidin-1-yl)phenyl)urea 48 (0.32 g, 30%)as light yellow
1
solid. H NMR (400 MHz, CDCl3) δ: 8.39 (s, 1H), 8.05 (d, J =
8.0 Hz, 2H), 7.63 (s, 1H), 7.46 (d, J = 4.0 Hz, 1H), 7.34 (s, 1H),
7.29-7.27 (m, 3H), 7.19-7.09 (m, 2H), 6.71-6.68 (m, 2H), 6.52 (d,
J = 4.0 Hz, 1H), 4.65 (d, J = 12.8 Hz, 1H), 4.40 (d, J = 12.8 Hz,
1H), 3.61-3.59 (m, 2H), 3.41-3.37 (m, 4H), 3.32-3.24 (m, 3H),
3.11-3.05 (m, 1H), 2.63-2.58 (m, 1H), 2.48-2.42 (m, 1H), 2.38 (s,
3H), 2.18-2.11 (m, 1H), 1.98-1.95 (m, 4H), 0.81 (s, 9H), -0.05 (s,
3H), -0.06 (s, 3H). 13C NMR (400 MHz, CDCl3) δ: 160.1, 157.7,
156.5, 154.6, 152.9, 146.7, 146.6, 145.4, 141.4, 139.0, 135.1,
129.7, 129.4, 128.2, 128.1, 128.0, 122.8, 121.8, 119.2, 119.0,
114.0, 113.8, 111.9, 111.8, 106.5, 106.3, 104.9, 104.8, 66.9, 61.5,
56.3, 53.3, 52.9, 50.2, 45.8, 29.7, 25.9, 25.2, 21.7, 18.3, -5.3, -
5.4; HR-ESI-MS (m/z): 812.3736, calcd for [M+ H]+
C42H54FN8O4SSi: 813.3737.
1-(2-Fluoro-6-(pyrrolidin-1-yl)phenyl)-3-(3-(1-(2-
hydroxyethyl)-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
2-yl)phenyl)urea (49). To a solution of 1-(3-(1-(2-((tert-
butyldimethylsilyl)oxy)ethyl)-4-(7-tosyl-7H-pyrrolo[2,3-
Cell-Based Assay: Human peripheral blood mononuclear cells
(PBMC) were purified from heparinized whole blood using
Ficoll-Paque (GE Healthcare Life Sciences, Pittsburgh, PA)
density gradient centrifugation. B cells were then enriched from
the isolated PBMC using the Human B Cell Isolation Kit II
(Miltenyi Biotec, Bergisch Gladbach, Germany) according to the
manufacturer’s instruction. A minimum purity of B cells (98%)
was confirmed by flow cytometry with an antibody against
human CD19 (BD Biosciences, Franklin Lakes, NJ). Purified B
cells (5 x 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.
d]pyrimidin-4-yl)piperazin-2-yl)phenyl)-3-(2-fluoro-6-
(pyrrolidin-1-yl)phenyl)urea 48 (0.32 g, 0.4 mmol, 1.0 eq) in
MeOH (10 mL) was added 12 N HCl (1 mL) at rt. The mixture
was stirred at rt for 2 h, then adjusted to pH = 7-8 with 3 N
NaOH and concentrated in vacuo. The residue (0.21 g, 0.32
mmol, 1.0 eq) was dissolved in MeOH/H2O (3:1 (v/v), 16 mL)
and treated with K2CO3 (0.27 mg, 1.92 mmol, 6.0 eq) while being
stirred at 65oC for 2 h. The mixture was adjusted pH = 7 with 3 N
HCl, concentration in vacuo to afford a residue which was
purified by prep-HPLC (MeOH/H2O with 0.05% NH4OH as
mobile phase from 5% to 95%) to afford 1-(2-fluoro-6-
(pyrrolidin-1-yl)phenyl)-3-(3-(1-(2-hydroxyethyl)-4-(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-2-yl)phenyl)urea 49 (0.12
1
g, 68%) as a white solid. H NMR (400 MHz, DMSO-d6) δ:
11.74 (s, 1H), 8.85 (s, 1H), 8.16 (s, 1H), 7.52-7.44 (m, 1H), 7.27-
7.02 (m, 4H), 6.55-6.50 (m, 3H), 4.69-4.58 (m, 2H), 4.39-4.37
(m, 1H), 3.46-3.26 (m, 9H), 3.00-2.94 (m, 1H), 2.54-2.50 (m,
1H), 2.37-2.35 (m, 1H), 2.07-2.05 (m, 1H), 1.87-1.85 (m, 4H).
13C NMR (400 MHz, DMSO-d6) δ: 162.0, 159.6, 156.3, 154.3,
152.5, 151.1, 149.1, 149.0, 142.3, 140.9, 129.4, 128.0, 127.9,
122.1, 121.4, 117.6, 117.5, 112.9, 112.7, 110.8, 104.4, 104.2,
102.6, 101.0, 67.0, 59.1, 56.8, 53.1, 52.7, 50.2, 46.2, 25.6; HR-
ESI-MS (m/z): 545.2792, calcd for [M+ H]+ C29H34FN8O2:
545.2783.
(R)
1-(2-Fluoro-6-(pyrrolidin-1-yl)phenyl)-3-(3-(1-(2-
hydroxyethyl)-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-
2-yl)phenyl)urea (50).
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
Preparative chiral SFC separation of 50 using the AS-H
(2 mm × 25 cm). Conditions: 50% methanol (0.1% DEA)/CO2 at
60 mL/min, 254 nm detection, 100 bar outlet pressure.
Approximately 78 mg of 49 (dissolved in 2 mg/mL, 1:1
MeOH/DCM) was purified to afford 50 (31 mg, rt 2.59, chemical
purity >99%, ee >99% as determined using analytical method:
AS-H (25 x 0.46 cm), 50% methanol (DEA)/CO2, 100 bar, 3
mL/min, 220 and 254 nm). The structure assignment for
compound 50 as determined using 1D proton, 2D HMQC,
HMBC and COSY experiments is available in the supplementary
material.
Methods for BTK biochemical activity assay: Compound
inhibition was measured after monitoring the amount of

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permeability coefficient, Papp, was calculated as follows:
Papp = (dCr /dt) x Vr / (A x CE)
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.
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Acknowledgments
The team is grateful for Biogen Inc. and Sunesis
Pharmaceuticals, Inc. for providing financial support to this
work.
BTK/ITK/TEC/TXK family located in chromosome Xp22.2.
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Supplementary data to this article can be found online at
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