Discovery of highly potent and selective Bruton's tyrosine kinase inhibitors: Pyridazinone analogs with improved metabolic stability

Article abstract of DOI:10.1016/j.bmcl.2015.11.076

BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of the acyclic amide bond connecting the terminal tetrahydrobenzothiophene

Full text of DOI:10.1016/j.bmcl.2015.11.076

Accepted Manuscript
Discovery of Highly Potent and Selective Bruton’s Tyrosine Kinase Inhibitors:
Pyridazinone Analogs with Improved Metabolic Stability
Wendy B. Young, James Barbosa, Peter Blomgren, Meire C. Bremer, James J.
Crawford, Donna Dambach, Charles Eigenbrot, Steve Gallion, Adam R.
Johnson, Jeffrey E. Kropf, Seung H. Lee, Lichuan Liu, Joseph W. Lubach, Jen
Macaluso, Pat Maciejewski, Scott A. Mitchell, Daniel F. Ortwine, Julie Di
Paolo, Karin Reif, Heleen Scheerens, Aaron Schmitt, Xiaojing Wang, Harvey
Wong, Jin-Ming Xiong, Jianjun Xu, Christine Yu, Zhongdong Zhao, Kevin S.
Currie
PII:
S0960-894X(15)30284-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.11.076
BMCL 23332
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 August 2015
17 November 2015
20 November 2015
Please cite this article as: Young, W.B., Barbosa, J., Blomgren, P., Bremer, M.C., Crawford, J.J., Dambach, D.,
Eigenbrot, C., Gallion, S., Johnson, A.R., Kropf, J.E., Lee, S.H., Liu, L., Lubach, J.W., Macaluso, J., Maciejewski,
P., Mitchell, S.A., Ortwine, D.F., Paolo, J.D., Reif, K., Scheerens, H., Schmitt, A., Wang, X., Wong, H., Xiong, J-
M., Xu, J., Yu, C., Zhao, Z., Currie, K.S., Discovery of Highly Potent and Selective Bruton’s Tyrosine Kinase
Inhibitors: Pyridazinone Analogs with Improved Metabolic Stability, Bioorganic & Medicinal Chemistry Letters
(2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.076
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Bioorganic & Medicinal Chemistry Letters
Discovery of Highly Potent and Selective Bruton’s Tyrosine Kinase Inhibitors:
Pyridazinone Analogs with Improved Metabolic Stability
Wendy B. Young*a, James Barbosab, Peter Blomgrenb, Meire C. Bremera, James J. Crawforda, Donna
Dambacha, Charles Eigenbrota, Steve Gallionc, Adam R. Johnsona, Jeffrey E. Kropfb, Seung H. Leeb,
Lichuan Liua, Joseph W. Lubacha, Jen Macalusob, Pat Maciejewskib, Scott A. Mitchellb, Daniel F. Ortwine
a, Julie Di Paolob, Karin Reif a, Heleen Scheerensa, Aaron Schmittb, Xiaojing Wanga, Harvey Wonga, Jin-
Ming Xiongb, Jianjun Xub, Christine Yu, Zhongdong Zhaob and Kevin S. Currieb
a Genentech, 1 DNA Way, South San Francisco, CA 94080
bGilead Sciences, 36 East Industrial Rd., Branford, CT 06405
cSt. Andrews Circle, Wallingford, CT 06492
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in
a suspension of clinical trials. The primary route of metabolism was through cleavage of the
acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker
aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted
in the identification of several central aryl linker substituents that conferred improved stability.
The most promising substituted aryl linkers were then incorporated into an optimized
pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved
potency, metabolic stability and preclinical properties.
Keywords:
Kinase inhibitor, Bruton’s tyrosine kinase, Btk,
Rheumatoid arthritis, pyridazinone
2009 Elsevier Ltd. All rights reserved.
Bruton’s tyrosine kinase (BTK) plays a critical role in the
development, differentiation and proliferation of B-lineage cells,
making it an attractive target for the treatment of immunological
disorders such as rheumatoid arthritis (RA), lupus, and multiple
sclerosis (MS)1-4, as well as B-cell malignancies.5 As outlined
in the preceding papers,6-7 we identified GDC-0834 (1) as a
highly potent and selective inhibitor of BTK with acceptable
pharmacokinetic (PK) and safety profiles in preclinical species.
Initially, we pursued two strategies to improve the metabolic
stability of the lead compound 1. The first was to remove the labile
acyclic amide entirely and replace it with common isosteres or
alternate linkers such as –NHSO2-, -SO2NH-, -NHCHCF3-, -
CONH-, and -NMeCO-. Unfortunately, all such modifications on
the GDC-0834 (1) scaffold or earlier tool molecules produced
analogs with dramatically reduced potency, likely due to a change
in the ligand conformation or disrupting the interaction with K430,6
so this endeavor was quickly abandoned. The second strategy
involved adding steric bulk around the acyclic amide to potentially
hinder amide cleavage. At the time of these medicinal chemistry
explorations we did not know what enzyme was responsible for
cleaving the amide bond, so a structure-based design approach
could not be applied.8 Therefore, we synthesized a range of analogs
In a clinical trial, 1 was found to be labile at the acyclic
amide bond resulting in rapid metabolism.7 The major metabolic
liability was identified to be cleavage of the primary amide to
liberate the corresponding aniline and acid by-products (Figure
1), making 1 unsuitable for further clinical developmentdue to
the low exposure of parent drug. This cleavage was determined
to result from a non-CYP mediated process that was more
prevalent in man than preclinical species such as mouse, rat,
dog, and monkey. Herein, we outline the discovery of second
generation pyridazin-3(2H)-one analogs with improved non-cyp
mediated metabolic stability, and which maintained the potency,
high target selectivity, and favorable safety profile of the original
series.

with mono- or bis- substitution ortho to the amide moiety linking
the central aryl to the 4,5,6,7-tetrahydrobenzo[b]thiophene group
(Table 1) to probe steric and electronic influences on this undesired
metabolic pathway. The goal was to find an analog closely related
to 1 that maintained many of its positive attributes (potency,
selectivity, safety profile), but was significantly less prone to
cleavage at the amide bond.
were within 4-fold of 1, showing that this region could tolerate
a variety of substituents. Pleasingly, several analogs (isomeric
pairs 7/8, 10/11, 13/14, and 15/16) had improved amide stability
and potency comparable to GDC-0834. Unfortunately, all these
analogs had minimal to no oral bioavailability in rats and hence
were not progressed further.
Table 1. Enzyme activity and in vitro stability SAR of BTK
inhibitors with central aryl group modifications
[
BTK
HLM (-) b,c,d,e
Confa
CMPD
X
Y
IC50 (nM)
Figure 1: The major metabolic pathway of GDC-0834 (1) in
human was determined to be amide cleavage to liberate by-
products M1 and M2.
1
2
Me
H
H
H
R
14
6
L
L
Rac
3
F
H
Rac
8
L
4
5
6
7
8
Cl
H
H
H
H
H
CN
Cl
F
F
Cl
Rac
Rac
Rac
U
U
Rac
6
1400
61
24
9
L
S
M
S
M
M
M
For synthetic expediency, we chose to generate the majority
of target compounds as racemic mixtures at the attachment
point of the piperazinone to the aryl group and then isolate the
enantiomers of selected compounds using supercritical fluid
chromatography (SFC). In a few cases, we synthesized the
individual (R) enantiomer directly as the required chiral
intermediate was available from the synthesis of GDC-0834.6
In the case of 15, we were able to assign the (S) configuration
after 16 (R) was unambiguously synthesized and a racemic
mixture of 15 and 16 was resolved by SFC. The sense of
chirality of each analog is noted in Table 1. Inhibition of the
target enzyme (BTK) was assessed and, for later examples
(Tables 2 and 3), the downstream effect on anti-IgM-stimulated
cell surface CD86 expression on B-cells. Cleavage of GDC-
0834’s amide bond was determined to be a non-CYP mediated
metabolic reaction based on its lack of NADPH dependence.7
In addition, metabolites associated with amide bond cleavage
were absent in in vitro incubations with purified CYPS serving
to confirm that CYPS were not involved. Therefore, metabolic
stability was monitored using in vitro human liver microsomes
in the absence of NADPH since the original amide cleavage
was determined not to be CYP-mediated and thus NADPH
independent.7 Performing the experiment in this manner
produced predominantly by-products of amide cleavage (e.g.
amines and acids, as depicted in Figure 1) and proved useful to
track the SAR of this undesired metabolism. Metabolism ID
studies were run after incubation with human liver microsomes
(in the absence of NADPH) to confirm by HPLC/MS that the
by-products were predominantly from amide cleavage.
Metabolism results are shown in Table 1 as categories (stable,
moderate, labile) for ease of comparison. Full in vitro predicted
hepatic clearance details can be found in the Supplementary
Materials.
9
10
Me
F
88
4
F
U
11
F
F
U
3
M
12
13
14
Cl
Me
Me
Cl
F
F
Rac
U
U
15
17
14
M
M
S
15
16
17
18
19
F
F
F
F
Cl
Me
Me
Cl
Cl
Me
S
R
U
U
U
6
6
23
9
S
S
S
S
S
39
20
21
Cl
Me
Me
U
5
S
S
Me
Rac
20
a) Configuration at the attachment point of the piperazinone to the aryl
group: Rac (racemate); (single stereoisomer unconfirmed
stereochemistry); (R configuration); S (S configuration); b) assay
conditions supplied in Supplementary Materials c) Human liver microsome
stability assay run in the absence (-) of NADPH; d) Metabolic Clearance
(CL) values (mL/min/kg): S (Stable) <6; M (Moderate) =6-15; L (Labile)
>15; e) The predicted hepatic clearance values are provided in the
Supplementary Materials.
U
R
At this point, we abandoned the concept of identifying a
close-in analog of 1 that would be candidate quality and instead
chose to re-tool the scaffold. We had identified several aryl
substitution patterns with potential to reduce cleavage of the
amide bond that could be incorporated into a next round of
designs. Along with continuing to probe aryl substitution, we
turned our attention to probing additional interactions between
the ligand and BTK protein to improve binding potency. We
looked to modify the methylpyrazin-2(1H)-one with alternate
heterocycles that could offer another H-bonding opportunity
with the protein. Amino-pyridazinones have been reported to
be efficient kinase hinge binders9 that have the potential to form
three H-bonds with the hinge region. Additionally, from
ongoing work in a related chemical series,10 we had discovered
that a 5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine in the
The baseline compound (2), lacking substitution at the 2- and
6- position, was labile at the amide bond. Mono ortho
substituted analogs 1, 3, and 4 were also highly labile,
indicating that steric bulk at the 2- position did not deter amide
cleavage. In contrast, substitution at the 6-position, as in
compounds 5-8, did inhibit cleavage and displayed improved
stability when compared to 1. Not surprisingly, compounds
with 2,6-disubstitution (9-21) were also more stable than 1.
Biochemical potencies of most analogs (except 5, 6, and 9)

solvent exposed region improved biochemical potency over the
dimethyl-2-piperazinone group within 1, and that oxidation of
the tetrahydrobenzo[b]thiophene to a benzothiophene ring
offered improved metabolic stability, in most analogs, as well.
Thus, with these changes in mind, we constructed a series of
second generation pyridazin-3(2H)-one analogs with selected
compounds as outlined in Table 2.
Table 3: Potency and metabolic stability of H3-substituted
pyridazinone BTK inhibitors
Table 2: Potency and metabolic stability of central aryl ring
substituted pyridazinone BTK inhibitors
IC50 nMa
HLM
(- / +)a,b,d,e
HHEP
X
CMPD
a,c,d,e
BTK
CD86
23
3
91
M / M
S
28
29
132
680
740
M / M
M
IC50 nMa
HLM
CMPD
X
Y
HHEP
(- / +)a,b,d,e
BTK
CD86
a,c,d,e
103
4
M / M
M / L
M / M
--
M
M
22
23
24
Me
Me
F
H
F
F
4
3
1
41
91
18
L / L
M / M
-- / L
--
S
M
30
31
54
90
25
Me
Me
20
387
S / L
--
4
26
27
F
H
Me
F
11
10
200
50
M / M
S / L
--
L
a) assay protocol in Supplementary Materials; b) human liver microsome
stability assay run in the presence (+) and absence (-) of NADPH; c) hHEP
= human liver hepatocyte stability d) Metabolic CL values (mL/min/kg): S
(stable) <6; M (moderate) = 6-15; L (labile) >15; e) The predicted
hepatic clearance values are provided in the Supplementary Materials.
a) assay protocols in Supplementary Materials; b) HLM=human liver
microsome stability assay run in the absence (-) and presence (+) of
NADPH; c) hHEP = human liver hepatocyte stability; d) Metabolic CL
values (mL/min/kg): S (stable) <6; M (moderate) = 6-15; L (labile) >15; e)
The predicted hepatic clearance values are provided in the
Supplementary Materials.
To assist in rationalizing the observed SAR and in the design
of novel congeners, a 1.36-Å resolution crystal structure of 23
in complex with BTK was obtained (Figure 3a). Interestingly,
although nearly every ring was modified from the original lead
GDC-0834 (1), 23 had a near identical binding mode (Figure
3b). The pyridazinone carbonyl and attached anilinic NH form
the expected acceptor/donor H-bonds with the backbone NH
and carbonyl of M477, holding the core heterocycle in place. A
third water-mediated H-bond is apparent between the
pyridazinone NH moiety and E475. The pyridazinone imino
ring nitrogen is adjacent to a water-filled cavity, and forms a
water mediated H-bond to K430. Thus, the polar face of the
pyridazinone occupies a polar internal area of the BTK protein,
contributing to the high binding affinity observed for this series.
In a similar vein to GDC-0834, the acyclic amide is rotated 78o
out of plane relative to the linker phenyl, and its carbonyl
accepts an H-bond from K430, although at an oblique angle.
The benzothiophene ring occupies an induced fit hydrophobic
site (“H3 site”) formed by D521, N526, D539, L542, S543, and
importantly, Y551 from the activation loop. This tyrosine
moves 17 Å from an observed apo orientation to encapsulate the
terminal phenyl ring of the benzothiophene.3 Because 23
contains no freely rotatable bonds and thus has low entropy, this
allows Y551 within the normally flexible activation loop to
“discover” the inhibitor terminal phenyl ring and pack against
it, effectively freezing the activation loop in an inactive
conformation. This binding mode undoubtedly contributes to
the exquisite sensitivity of this ring to substitution. The 5-
In this series, 22 represents a baseline compound with the
central aryl ring maintaining the same substitution pattern at the
2- and 6- positions as the original lead 1. In comparison to 1,
22 offered a moderate improvement in BTK biochemical and
cell potency (1, BTK IC50 = 14 nM; CD86 IC50=60 nM) but no
improvement in overall metabolic stability. We were pleased to
find that when some of the preferred central aryl ring
substituents explored in the pyrazinone series (Table 1) were
applied to these new pyridazinones, improvements in metabolic
stability were seen as compared to the baseline pyridazinone 22.
All central aryl substituted analogs (23, 25-27) except 24,
showed an improvement in metabolic stability in human liver
microsomes in the absence of NADPH. Of these aryl-
substituted analogs, only 23 and 26 offered moderate stability in
liver microsomes in the presence of NADPH (as compared to
22). In addition, 23 was found to be stable in human liver
hepatocytes. Thus, of all these compounds, 23 had the best
overall profile with moderate stability in human liver
microsomes (-/+NADPH), low clearance in human hepatocytes,
and acceptable BTK biochemical and cell potency.
We next probed replacements of the benzothiophene group
with isosteres and/or other aryl groups that we found suitable
from earlier work.6 Key analogs are shown in Table 3. Aza-
analogs of the benzothiophene, as in 28 and 29, dramatically
decreased affinity for BTK, presumably due to incompatibility
with the lipophilic nature of the binding pocket. Altering the
aryl group to two of our earlier and more potent
monosubstituted aromatic rings,6 as in 30 and 31, offered potent
and moderately stable compounds. Unfortunately these
compounds lacked oral bioavailability in rat. Thus, 23 was
determined to be the preferred compound to progress.
methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine
largely
extends into solvent, making contact with a portion of Y476.

in murine B cells with an EC50 of 91 nM. Additionally, 23
strongly inhibited anti-IgM stimulated B-cell proliferation
(EC50=6 nM), and immune-complex (IC) triggered cytokine
production by human monocytes (EC50 = 37 nM [TNFα]; EC50
= 22 nM [IL1]). Consistent with the lack of Btk expression in
T cells and its selectivity profile, 23 did not affect T-cell
proliferation (EC50> 5,000 nM).
(a)
Table 4: Potency and selectivity of 23 versus other kinases with
>50% inhibition at 1 uM
BTK BMX TEC FGR
SRC
Kinase
MELK
3
14
5x
230
77x
1033 >10000
344x >3333x
924
IC50 (nM)
---
308x
fold vs BTK
Table 5: Whole Blood potency, human B-cell proliferation and
monocyte cytokine production of 23
(b)
Assay
EC50 (nM)
Whole blood CD63
257
Human B-cell proliferation
Human T-cell proliferation
IC-triggered TNF release from monocytes
IC-stim IL-1 release from monocytes
6
>5000
37
22
The predicted human clearance of 23 was moderate (12
mL/min/kg) and low (5 mL/min/kg) as respectively determined
by liver microsome and hepatocyte assessments (Table 6). The
in vivo clearances were moderate in mouse (43 mL/min/kg) and
dog (14 mL/min/kg), and low in rat (5 mL/min/kg). Oral
bioavailabilities from solution dosing ranged from a low of 26%
(mouse) to a high of 65% (dog). Notably, the in vivo clearances
in mouse, rat and dog more closely aligned with the in vitro
hepatocyte clearance data, providing increased confidence that
the in vivo human clearance would be consistent with the lower
in vitro hepatocyte values rather than to the higher liver
microsome values.
Figure 3. (a) Crystal structure of 23 complexed to BTK; (b)
Superposition of GDC-0834 (green) and 23 (orange) from their
protein/ligand X-ray structures after aligning the proteins.
Importantly, 23 demonstrated
excellent biochemical
selectivity when screened at 1 uM against a panel of 228
kinases in the SelectScreen® Kinase Profiling Services of
Invitrogen-Life Technologies (Madison, USA), showing >50%
inhibition of the activity of only 6 kinases - BTK, BMX, TEC,
FGR, MELK, and SRC (data in Supplementary Material). We
obtained IC50 values for these kinases to increase the precision
of the selectivity profile (as shown in Table 4). Compound 23
had greater than 300-fold selectivity for BTK versus FGR, SRC
and MELK, thus providing a low risk of inhibiting these kinase
targets. The margins of 23 against BMX and TEC were
smaller, with only 5-fold and 77-fold selectivity, respectively.
To our knowledge, there are no reports of clinical safety data
associated with potent and selective inhibitors of BMX or TEC
kinases to determine the risk of inhibiting these off-targets.
However, existing non-clinical data related to such inhibition
have not been cause for concern.11-14 In the case of TEC,14
inhibition of this protein may in fact have a beneficial effect as
an anti-inflammatory agent.
Table 6: Preclinical DMPK Profile of 23
Preclinical DMPK Data
Predicted hepatic clearance
(mL/min/kg)
Fa, b
Species
in vitro
in
vivoc
liver
liver
microsomes
hepatocytes
mouse
rat
56
25
24
22
12
42
17
15
16
4
43
5
34%
26%
65%
--
Further profiling indicated that 23 had no significant off-
target activity when screened against a panel of 30 receptors (all
< 35% at 10 uM). Only minimal inhibition of the hERG
channel (27% at 10 uM) was found, and 23 was negative in
gene-tox profile Ames (TA98/100 strains; +/- S9) and
micronucleus tests (+/- S9).
dog
14
4
cyno
human
aF = oral bioavailability after a 5 mg/kg dose (n=3)
b 60%PEG400/ 15% EtOH/ 50 mm Citrate buffer (pH 4.2)c
Additional in vitro potency data of 23 is detailed in Table 5.
In human whole blood, 23 demonstrated potent inhibition of
anti-IgE stimulated CD63 expression on basophils, with an EC50
of 257 nM. Furthermore, 23 inhibited cellular functions in
murine and human B cells, as well as human monocytes.
Specifically, CD86 induction was reduced after BCR activation

Scheme 1: Synthesis of 23
their contributions in generating the data for this manuscript.
Use of the Stanford Synchrotron Radiation Lightsource, SLAC
National Accelerator Laboratory, is supported by the U.S.
Department of Energy, Office of Science, Office of Basic
Energy Sciences under Contract No. DE-AC02-76SF00515.
References and notes
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J.K.; Le, S.H.; Liu, L.; Lubach, J.; Macaluso, J.; Maciejewski, P.;
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accepted.
The synthesis of 23 is shown in Scheme 1 (details in
Supplementary Materials). Starting from 4-bromo-6-
chloropyridazin-3(2H)-one, Pd-catalyzed C-N coupling (I) with
5-Methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine (II)
gave the coupled product (III) in 74% yield. Compound III
was then converted to 23 via a Suzuki–Miyaura coupling with
IV, in 50% yield.
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Baumgardner, M.; Le, H.; Gopaul, S.; Boggs, J.; Kuebler, P.; Davis,
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specie (data not shown). However, a re-design around this
scaffold led to the identification of a more potent and stable
pyridazinone hinge-binding motif with appreciable oral
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selected as the lead for further profiling.
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Acknowledgments
We thank the BTK team members at Gilead (formerly CGI
Pharmaceuticals Inc.), Genentech, ChemPartner, and AMRI for

Graphical Abstract
Discovery of Potent and Selective Bruton’s Tyrosine
Kinase Inhibitors: Pyridazinone Analogs with
Improved Metabolic Stability
Leave this area blank for abstract info.
Wendy B. Young, James Barbosa, Peter Blomgren, Meire C. Bremer, James J. Crawford, Donna Dambach, Charles Eigenbrot,
Steve Gallion, Adam R. Johnson, Jeffrey E. Kropf, Seung H. Lee, Lichuan Liu, Joseph W. Lubach, Jen Macaluso, Pat
Maciejewski, Scott A. Mitchell, Daniel F. Ortwine, Julie Di Paolo, Karin Reif, Heleen Scheerens, Aaron Schmitt, Xiaojing Wang,
Harvey Wong, Jin-Ming Xiong, Jianjun Xu, Christine Yu, Zhongdong Zhao and Kevin S. Currie