Discovery of Potent and Selective Tricyclic Inhibitors of Bruton's Tyrosine Kinase with Improved Druglike Properties

Article abstract of DOI:10.1021/acsmedchemlett.7b00103

In our continued effort to discover and develop best-in-class Bruton's tyrosine kinase (Btk) inhibitors for the treatment of B-cell lymphomas, rheumatoid arthritis, and systemic lupus erythematosus, we devised a series of novel tricyclic compounds that improved upon the druglike properties of our previous chemical matter. Compounds exemplified by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated, and efficacious in an animal model of arthritis.

Full text of DOI:10.1021/acsmedchemlett.7b00103

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Letter
Discovery of Potent and Selective Tricyclic Inhibitors of
Bruton’s Tyrosine Kinase with Improved Drug-like Properties
Xiaojing Wang, James Barbosa, Peter Blomgren, Meira C. Bremer, Jacob Chen, James J. Crawford, Wei
Deng, Liming Dong, Charles Eigenbrot, Steve Gallion, Jonathan Hau, Huiyong Hu, Adam R. Johnston, Arna
Katewa, Jeffrey E. Kropf, Seung H. Lee, Lichuan Liu, Joseph W. Lubach, Jen Macaluso, Pat Maciejewski,
Scott Allan Mitchell, Daniel Fred Ortwine, Julie DiPaolo, Karin Reif, Heleen Scheerens, Aaron Schmitt, Harvey
Wong, Jin-Ming Xiong, Jianjun Xu, Zhongdong Zhao, Fusheng Zhou, Kevin S. Currie, and Wendy B. Young
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 03 May 2017
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Discovery of Potent and Selective Tricyclic Inhibitors of Bruton’s Tyro-
sine Kinase with Improved Drug-like Properties
Xiaojing Wanga *, James Barbosab, Peter Blomgrenb, Meire C. Bremera, Jacob Chena, James J. Craw-
forda, Wei Dengc, Liming Dongc, Charles Eigenbrota, Steve Gallionb, Jonathon Haua, Huiyong Hua, Ad-
am R. Johnsona, Arna Katewaa, Jeffrey E. Kropfb, Seung H. Leeb, Lichuan Liua, Joseph W. Lubacha, Jen
Macalusob, Pat Maciejewskib, Scott A. Mitchellb, Daniel F. Ortwinea, Julie DiPaolob, Karin Reifa,
Heleen Scheerensa, Aaron Schmittb, Harvey Wonga, Jin-Ming Xiongb, Jianjun Xub, Zhongdong Zhaob,
Fusheng Zhouc, Kevin S. Currieb and Wendy B. Younga *
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aGenentech, Inc., Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, United States
bGilead Sciences, 199 East Blaine St., Seattle, WA, 98102 United States
cChemPartner, No. 1 Building, 998 Halei Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai China, 201203.
KEYWORDS: Kinase inhibitor, Btk, Rheumatoid arthritis, Lupus, G-744.
ABSTRACT: In our continued effort to discover and develop best-in-class Bruton’s tyrosine kinase (Btk) inhibitors for the
treatment of B-cell lymphomas, rheumatoid arthritis and systemic lupus erythematosus, we devised a series of novel tri-
cyclic compounds that improved upon the drug-like properties of our previous chemical matter. Compounds exemplified
by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated and efficacious in an animal model of
arthritis.
Bruton’s tyrosine kinase (Btk) plays a critical role in the
function of B cells and myeloid cells, making it an attrac-
Table 1: Improved properties of tricyclic H3 Btk inhibitors
tive target for the treatment of immunological disorders
such as rheumatoid arthritis (RA), systemic lupus erythe-
matosus (SLE), multiple sclerosis (MS).1-4 as well as B-cell
lymphomas.5 Accordingly, there have been significant ef-
forts from the pharmaceutical community toward identify-
ing Btk inhibitors for clinical evaluation.6-14 Of these, the
most advanced compound to date is ibrutinib, recently
approved for treatment of Mantle Cell Lymphoma (MCL),
chronic lymphocytic leukemia (CLL), and Waldenstrom’s
macroglobulinemia and under evaluation in additional
indications.15
ID
IC50 (μM)a
LLEb hPPBc
Rat
Btk
0.042
0.011
CD86
0.74
--
CLd
2
--
F%e
45%
--
1
2a
3.6
4.2
5.8
99%
99%
97%
We have previously reported on the discovery of several
series of novel and potent Btk inhibitors that possess ex-
quisite selectivity for Btk over other kinases.9-11,16 Their high
potency and selectivity stems from the ability of com-
pounds such as 1 to create an induced binding fit in the
protein via a rearrangement of the activation loop.3 Other
kinases, including members of the tyrosine kinase family
cannot rearrange in the exact same way due to differences
in the amino acid sequence within this loop.11 The tetrahy-
drobenzothiophene moiety within 1, held rigidly in place
by other contacts the inhibitor makes with the protein,
presents a hydrophobic surface that “attracts and se-
questers” Y551, ultimately creating a lipophilic specificity
pocket (“H3”). A common feature in this series is the in-
corporation of a secondary amide that tethers the distal
bicyclic ring to the central benzene. We set out to address
metabolism and permeability liabilities associated with the
amide moiety.
2b 0.001
0.087
2
90%
aAssay protocols in Supporting Information (SI), n > 2. bLLE =
pIC50(Btk) – cgLogD. Human plasma protein binding. dTotal clearance
c
(mL/min/kg) at 1 mg/kg i.v. dose formulated using a mixture of
EtOH/Cremophor/water for 1 (solution) or PEG400/EtOH/water for 2b
(solutions). F% = oral bioavailability after a 5 mg/kg oral dose (n=3)
formulated using a mixture of EtOH and Cremophor for 1 (solution) or
PEG400/EtOH/Tween80/water for 2b (suspension).
e
In particular, we hypothesized that molecules with an am-
ide tethered back onto the tetrahydrobenzothiophene,
such as 2a, would offer a molecule with one less exposed
N-H donor and reduce the number of rotatable bonds,
potentially improving potency and permeability. Crystal
structures of related compounds bound to Btk indicated
the amide N-H was not interacting with the protein.3 Ad-
ditionally, we incorporated a hydroxyl group in the central
benzene ring that was well positioned to interact with a
nearby K430 and D539 residues, with the hope of improv-
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that the most potent compounds 2b and 3 (Btk IC50
ing binding affinity. Table 1 shows the potencies of these
=
compounds for Btk in a biochemical assay, as well as their
potencies in an in vitro mouse splenocyte CD86 cell based
assay that was used as a downstream pharmacodynamic
marker for Btk activity (assay protocols in SI). Pleasingly,
the newly generated tricyclic compound 2a was roughly 4-
fold more potent against Btk than the uncyclized precur-
sor 1. Compound 2b, which includes a hydroxyl group on
the central benzene ring, has a ~10-fold increase in Btk
binding potency than 2a and is roughly 40-fold more po-
tent than compound 1. With the improved potency and/or
decreasing lipophilicity, the lipophilic ligand efficiency
(LLE) increased sequentially from 1 (3.6) to 2a (4.2) to 2b
(5.8), indicating enhanced drug-like properties. Cellular
potency of compound 2b (IC50 = 0.087 M), as measured
by inhibition of CD86 surface expression on B cells, is 8-
fold higher than 1 (IC50 = 0.74 M). Additionally, 2b is 2-
fold more orally bioavailable (90%) than 1 (45%) and has
similarly low clearance in rat.
0.001, 0.006 M, respectively) contain all-carbon fused
cyclohexane rings.
1
2
3
4
5
6
7
8
Table 2: SAR for H3 groups
ID
R
IC50 (μM)a
S
9
(M)b
Btk
CD86
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2b
3
0.001
0.006
0.081
2.6
0.087
2
0.2
49
85
36
124
4
0.48
Figure 1: Superposition of modeled structures of 1 (ma-
genta) and 2b (cyan) in the Btk catalytic domain. H-bonds
to the protein are shown as black cylinders with dashed
lines. The active site surface is shown color coded by lipo-
philicity (green, nonpolar; purple, polar).
5
48.6
>50
6
16.7
7
0.002 0.064
0.002 0.057
1.4
1
G-744
8
9
-
0.051
2
10
11
0.003
0.139
0.193
NT
2
56
12
13
14
0.003
0.004
0.005
1.2
8
15
2
Modeling of 1 and 2b in the Btk catalytic domain is shown
in Figure 1. The tricyclic system did not alter the overall
predicted ligand binding mode, and preserved the interac-
tions at the kinase selectivity H3 pocket with the neighbor-
ing hydrophobic residues. Specific interactions with Y551
and K430 were maintained. The hydroxymethyl on the
central benzene ring projected into a water-filled cavity,
and formed H-bonds with K430 and D539.
0.171
0.23
aAssay protocol in SI, n > 2. bKinetic solubility was measured at pH7.4.17
The success of our initial attempts at creating improved
Btk inhibitors by the employment of a tricyclic moiety en-
couraged us to explore additional tricycles at this position
as well as carefully designed bicycles. Table 2 shows a se-
lected subset of such compounds. Compounds 2b-6 are
examples of inhibitors that contain distal 6-5-6 tricyclic
ring systems. Since the H3 site is lipophilic, with nonpolar
residues flanking much of this pocket, it is not surprising
Compound 4, with a heteroatom in the right hand saturat-
ed ring, displayed reduced potency (Btk IC50 = 0.081 M),
while 5 and 6 were much less potent, presumably due to
the reduced lipophilicity of the tricycle. Modeling indicat-
ed that there was an opportunity to further flesh out the
H3 pocket and that gem dimethyl substituted 6-5-5 sys-
tems might offer some advantages by occupying more of
the lipophilic H3 pocket. The resulting 6-5-5 compounds 7
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and 8 of 2b and 3 respectively were equi- to slightly more
potent against Btk (IC50 = 0.002 M), and both had im-
proved cell potencies.
With a variety of novel tricyclic compounds in hand that
had excellent potency for Btk, we focused our efforts on
designing compounds with improved physiochemical
properties. As demonstrated in Table 2, although com-
pounds 2b, 7, 8, and 9 had the best cellular potency (CD86
IC50 < 100 nM), they unfortunately all had low kinetic solu-
bility (< 2 M) at physiological pH. Kinetic solubility as
measured in an internal high throughput assay17 was used
as an initial gauge of intrinsic solubility. Low solubility is a
primary cause of reduced oral bioavailability of the crystal-
line materials preferred for clinical development, and often
presents formulation challenges in dose escalations for
safety studies. This was indeed the case for many com-
pounds in this chemical series. To find compounds with
improved solubility, modifications of the left-hand portion
of the inhibitor that extends into a partially solvent ex-
posed area of the protein region (“H2”) were explored and
found to be well tolerated. Of the many compounds gen-
erated,21 we determined that compounds with a substitut-
ed pyridinopiperazine group (Table 3), as opposed to the
pyrimidine of the previous compounds (Table 2), offered
an exciting new sub-series with much improved kinetic
solubility.
1
2
3
4
5
6
7
8
We also examined bicyclic 6-6 fused (9), 5-5 fused (10-11),
and 5-6 fused (12) ring systems containing appropriately
placed t-butyl groups to extend into the H3 pocket.
Among this group, the 5-5, 5-6 fused 10 and 12 had the
highest enzyme binding potency (Btk IC50’s = 0.003 M)
but were relatively less active in the CD86 cellular assay.
9
Finally, we surveyed 7-6 ring systems (13 and 14). These
compounds were less appealing due to their decreased
biochemical and cell potencies. The cell potencies of all
compounds mostly tracked with biochemical potencies,
with the Btk/CD86 IC50 ratio ranging from 6- (4) to 64-
(10) fold. The only major outlier was the bicyclic com-
pound 12 which had a cell shift of 400-fold. Presumably,
this shift was due to differences in plasma protein binding
and cell permeability of this compound. Other researchers
have reported on a variety of bicyclic H3 moieties within
alternate series,18-20 however we are the first group to de-
scribe highly potent and Btk-selective tricyclic 6-5-6 and 6-
5-5 compounds.
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Table 3: Combination of the best H2 and H3 moieties for optimal potency and drug-like properties
IC50 (μM)a
Rat
HHep/
RHep b
Sol.
ID
X
Y
Z
Perm.d
(M)c
f
Btk
CD86
WB CD69
CLe
16
F%
41
7
H
H
n/a
Me
0.002
0.064
0.087
11 / <10
1.4
18
MOD
-
15
0.003
0.025
-
<6.2 / <10
29
37
16
17
H
H
H
F
ox
Me
ox
0.004
0.004
0.008
0.002
0.004
0.006
0.107
0.137
0.022
0.010
0.089
0.045
0.069
0.029
0.035
8.8 / <10
10 / 12
1
MOD
MOD
MOD
MOD
HIGH
-
-
115
99
71
40
14
37
20
20
46
52
27
18
19
20
<6 / <10
<6.2 / <10.1
Me
F
ox
<6 / <10
73
b
c
aAssay protocol in SI, n > 2. Projected hepatic clearance using human or rat hepatocytes. Kinetic solubility was measured at pH7.4;17 dMeasured
permeability using Madin-Darby Canine Kidney (MDCK) Epithelial cell lines, A to B; MOD: 1-10 (10-6 cm/s) and HIGH: > 10 (10-6 cm/s). eTotal clear-
ance (mL/min/kg) at 1 mg/kg i.v. dose formulated using a mixture of PEG400/ethanol/water for 7, 15, and 17-20 (solution); fF% = oral bioavailability
after 5 mg/kg oral dose (n=3) formulated using a mixture of PEG400/ethanol/Tween80/water for 7 (suspension), PEG400/ethanol/water for 17 (sus-
pension) and 15, 18-20 (solution).
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A small subset of compounds containing combinations of
activation with immune-complexes was abrogated by G-
the best H2 and H3 groups are highlighted in Table 3.
Installing a tertiary basic amine containing left-hand H2,
which is primarily protonated at neutral pH, indeed
helped improve the solubility from 1.4 M (7) to 18 M
(15). Reducing the basicity from calculated pKa of 7.8 (15)
to 6.3 (16, substituted with an oxetane)22 abrogated the
improved solubility. This prompted us to focus on H3
groups with higher polarity than that of 7. Gratifyingly, all
three compounds containing a more polar tetrahydroin-
dole H3 group (17-20) maintained high kinetic solubility
regardless of the amine basicity. However, the moderate
CD86 cellular potencies of 17 and 18 (IC50‘s of 0.107 and
0.137 M, respectively) mirrored that of the 0.2 M poten-
cy seen for 3, which bears the same polar H3 group. A
potency breakthrough was realized when a single fluorine
atom was added to the middle linker benzene ring (19-
20). This fluorine is well positioned to engage the back-
bone carbonyl carbon of G409 in a dipole-dipole interac-
tion (Figure s1 in SI), further stabilizing the binding orien-
tation of these inhibitors in the Btk catalytic domain. The
resultant CD86 cellular potency improved 5-fold and 13-
fold for the two pairs 1719 and 1820 respectively.
744 (EC50 = 33 nM). In human whole blood, G-744
demonstrated potent inhibition of BCR-stimulated CD69
expression on B-cells with an EC50 of 87 nM.
Table 4: Additional G-744 potency data (mean + SEM)
Assay
Btk biochemical, (Ki,app
Mouse splenocyte B cell CD86
Human Whole Blood B cell CD69
Human B cell proliferation
Ki,app or EC50 (nM)
1.28 + 0.13 [n = 3]
55 and 75 [n = 2]
87 + 30 [n = 11]
22 + 3 [n = 17]
9
)
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Human monocyte TNFα production
33 + 6 [n = 12]
In pharmacokinetic experiments, G-744 exhibited low to
moderate clearance in four preclinical species (Table 5).
Sufficient oral exposures were achievable using a crystal-
line formulation in both mouse and rat despite low kinet-
ic solubility.
Table 5: Preclinical DMPK profiling of G-744
Species
In vitro
In vivo
CLc F%d
22 45e - 77f
a
b
All compounds in table 3, except 17, were low to moder-
ately cleared and had reasonable bioavailability in in vivo
rat PK studies. In vitro hepatic clearance represented by
RHEP data underestimated the in vivo clearance, however
the relative stability trend was consistent from in vitro to
in vivo studies. It is also pleasing to see that human hepat-
ic clearance ranged from low to moderate. Given the to-
tality of data including safety assessment (not discussed
here), compounds 7 (G-744) and 20 rose to the top of the
list with the most balanced overall profile.
LM CLhep Hep CLhep
mouse
rat
35
18
15
18
8
2
3
16
7
23e - 37f
dog
1
27f
cyno
31
17
--
human
11
--
--
aProjected hepatic clearance using liver microsomes (mL/min/kg).
c
bProjected hepatic clearance using hepatocytes (mL/min/kg). Total
In addition to potency and drug-like properties, a promi-
nent goal of developing a clinically viable kinase inhibitor
lies in controlling selectivity. Poor selectivity may have a
profound effect on drug safety, especially for non-
oncology indications where chronic dosing requires an
exquisitely clean safety profile. Therefore both G-744 and
20 were profiled against a panel of 285 active recombi-
nant human kinases. In particular, G-744 demonstrated
>1000-fold Btk biochemical selectivity against all kinases
tested except for EphA7 and Fgr, against which it still
showed robust selectivity of 428-fold and 868-fold, re-
spectively (Figure 3s in SI). Due to G-744’s superb kinase
selectivity (superior to 20), we realized it could be an ex-
cellent tool molecule to probe the biology of Btk as the
results would not be confounded by off-target activity
and the need for interpretation. Thus, we performed a full
characterization of this molecule, with key data summa-
rized in Table 4. In addition to preventing cellular func-
tions in murine B-cells such as B-cell receptor (BCR)-
mediated CD86 induction with an EC50 of 64 nM, G-744
also inhibited BCR-stimulated B-cell proliferation in hu-
man B-cells (EC50 = 22 nM). In human moncocytes, pro-
duction of the inflammatory cytokine TNFα following
clearance in vivo (mL/min/kg). dF% = oral bioavailability after 5
(amorphous) or 100 mg/kg (crystalline) dose in mouse; 5 (amorphous)
or 100 mg/kg (crystalline) dose in rat; 5 mg/kg dose in dog (n = 3).
eMethylcellulose/Tween80/water suspension of crystalline free base
material. fHydroxypropylmethethylcellulose/Na citrate/water pH 3
suspension of amorphous material
Given the favorable DMPK profile of G-744 in rat, we ex-
amined its efficacy in the developing collagen-induced
arthritis (CIA) model in Lewis rat.3,23 Oral dosing with G-
744 at 6.25, 12.5 and 25 mg/kg b.i.d. overall maintained
plasma concentrations above the IC50, IC70 and IC90, re-
spectively, for inhibition of Btk Y223 phosphorylation in
whole blood (Figure s2 in SI). As shown in Figure 2, all
three doses resulted in a significant dose-dependent inhi-
bition of ankle thickness between day 10 and day 17 (onset
of increase in ankle diameter on day 9). The 25 mg/kg
dose (97% inhibition of the area under the ankle thick-
ness vehicle curve) showed comparable efficacy to dexa-
methasone. In naïve rats treated with vehicle, the ankle
diameters did not change over the course of the study and
ankles from normal rats were significantly different (P <
0.05) compared with the CIA rats treated with vehicle. In
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The manuscript was written with contributions by all au-
thors. All authors have given approval to the final version of
the manuscript.
addition, G-744 was also highly efficacious in preventing
IFNα-driven lupus nephritis.24
1
2
3
4
5
6
7
8
9
Funding Sources
Figure 2: Rat CIA Study - Treatment with G-744 protects
Lewis rats from collagen-induced arthritis. Female Lewis
rats (n = 10 per group) with developing CIA were dosed
orally with G-744 as indicated (b.i.d.) or 0.05 mg/kg dex-
amethasone daily starting on day 0. Daily ankle diameter
measurements are shown as mean ± SEM and were sig-
nificantly (by ANOVA) reduced toward normal for all
drug-treated rats (significant days 10-17) as compared to
the vehicle control.
Thanks to Genentech, a member of the Roche Group for the
research funds.
ACKNOWLEDGMENT
We are grateful to analytical, purification chemistry and
compound management group for compound characteriza-
tion, purification and handling.
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ABBREVIATIONS
Btk, Bruton’s tyrosine kinase; RA, Rheumatoid arthritis; SLE,
systemic lupus erythematosus; MS, Multiple sclerosis; MCL,
Mantle cell lymphoma; CLL, chronic lymphocytic leukemia;
BCR, B-cell receptor; DMPK, Drug metabolism and pharma-
cokinetics; CIA, collagen-induced arthritis; b.i.d., twice a day.
SEM: standard error of the mean.
Vehicle
6.25 mg/kg G-744
0.34
12.5 mg/kg G-744
0.33
25 mg/kg G-744
0.32
Dexamethasone
0.31
Naive
0.30
REFERENCES
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0.25
(1)
Satterthwaite, A. B.; Witte, O. N. The Role of Bruton's
Tyrosine Kinase in B-Cell Development and Function:
a Genetic Perspective. Immunol. Rev. 2000, 175, 120–
127.
(2)
(3)
Brunner, C.; Müller, B.; Wirth, T. Bruton's Tyrosine
Kinase Is Involved in Innate and Adaptive Immunity.
Histol. Histopathol. 2005, 20 (3), 945–955.
9
10 11 12 13 14 15 16 17
Day
Di Paolo, J. A.; Huang, T.; Balazs, M.; Barbosa, J.;
Barck, K. H.; Bravo, B. J.; Carano, R. A. D.; Darrow, J.;
Davies, D. R.; DeForge, L. E.; Diehl, L.; Ferrando, R.;
Gallion, S. L.; Giannetti, A. M.; Gribling, P.; Hurez, V.;
Hymowitz, S. G.; Jones, R.; Kropf, J. E.; Lee, W. P.;
Maciejewski, P. M.; Mitchell, S. A.; Rong, H.; Staker,
B. L.; Whitney, J. A.; Yeh, S.; Young, W. B.; Yu, C.;
Zhang, J.; Reif, K.; Currie, K. S. Specific Btk Inhibition
Suppresses B Cell- and Myeloid Cell-Mediated Arthri-
tis. Nat. Chem. Biol. 2011, 7 (1), 41–50.
In summary, orally bioavailable Btk inhibitors with novel
tricyclic head groups were discovered through structure
and property based drug design. Improved molecules
were discovered and an outstanding tool molecule, G-744,
was identified with excellent potency, favorable DMPK
properties and superb kinase selectivity. G-744 demon-
strated efficacy equivalent to Dexamethasone in a rat CIA
model at 25 mg/kg b.i.d. dosing. Such findings further
solidified our commitment to Btk as a therapeutic target.
The chemistry culminating in a clinical candidate will be
described in a subsequent manuscript.
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Puri, K. D.; Di Paolo, J. A.; Gold, M. R. B-Cell Receptor
Signaling Inhibitors for Treatment of Autoimmune
Inflammatory Diseases and B-Cell Malignancies. Int.
Rev. Immunol. 2013, 32 (4), 397–427.
Robak, T.; Robak, E. Tyrosine Kinase Inhibitors as
Potential Drugs for B-Cell Lymphoid Malignancies
and Autoimmune Disorders. Expert Opin. Investig.
Drugs 2012, 21 (7), 921–947.
Lou, Y.; Owens, T. D.; Kuglstatter, A.; Kondru, R. K.;
Goldstein, D. M. Bruton's Tyrosine Kinase Inhibitors:
Approaches to Potent and Selective Inhibition, Pre-
clinical and Clinical Evaluation for Inflammatory Dis-
eases and B Cell Malignancies. J. Med. Chem. 2012, 55
(10), 4539–4550.
ASSOCIATED CONTENT
Supporting Information (SI). Physiochemical properties,
experimental procedures, compound characterization, assay
protocols, PK-PD relationship, and full kinome data are in-
cluded. This information is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Currie, K. S. 2015 Medicinal Chemistry Reviews; Desai,
M. C., Ed.; 2016; pp 225–234.
Xing, L.; Huang, A. Bruton's TK Inhibitors: Structural
Insights and Evolution of Clinical Candidates. Future
Med. Chem. 2014, 6 (6), 675–695.
Corresponding Author
* email: young.wendy@gene.com (W. B. Young).
* email: wang.xiaojing@gene.com (X. Wang).
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Young, W. B.; Barbosa, J.; Blomgren, P.; Bremer, M.
C.; Crawford, J. J.; Dambach, D.; Eigenbrot, C.; Gal-
lion, 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.; Di Paolo, J.; 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 Inhibi-
ORCID
Xiaojing Wang: 0000-0003-1575-3227
Wendy Young: 0000-0003-4329-7580
Author Contributions
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ACS Medicinal Chemistry Letters
tors: Pyridazinone Analogs with Improved Metabolic
Stability. Bioorg. Med. Chem. Lett. 2016, 26 (2), 575–
579.
2015, 1358 (1), 82–94.
1
2
3
4
5
6
7
8
9
(16)
(17)
Blomgren, P. A.; Lee, S. H.; Mitchell, S. A.; Xu, J.;
Schmitt, A. C.; Kropf, J. E.; Currie, K. S. Certain Sub-
stituted Amides, Method of Making, and Method of
Use Thereof. US Patent Office US 2008/0153834 A1
June 26, 2008, pp 1–47.
Lin, B.; Pease, J. H. A High Throughput Solubility
Assay for Drug Discovery Using Microscale Shake-
Flask and Rapid UHPLC-UV-CLND Quantification. J.
Pharm. Biomed. Anal. 2016, 122, 126–140.
Dewdney, N. J.; Kondru, R. K.; Lou, Y.; Soth, M. Btk
Protein Kinase Inhibitors. US Patent Office US
2009/0105209 A1 April 2009.
Dewdney, N. J.; Lou, Y.; Soth, M. Novel Imidazo[1,2-
a]Pyridine and Imidazo[1,2-B]Pyridazine Derivatives.
World Intellectual Property Organization WO
2009/077334 A1 June 25, 2009, pp 1–118.
Lou, Y.; Han, X.; Kuglstatter, A.; Kondru, R. K.;
Sweeney, Z. K.; Soth, M.; McIntosh, J.; Litman, R.;
Suh, J.; Kocer, B.; Davis, D.; Park, J.; Frauchiger, S.;
Dewdney, N.; Zecic, H.; Taygerly, J. P.; Sarma, K.;
Hong, J.; Hill, R. J.; Gabriel, T.; Goldstein, D. M.; Ow-
ens, T. D. Structure-Based Drug Design of RN486, a
Potent and Selective Bruton's Tyrosine Kinase (BTK)
Inhibitor, for the Treatment of Rheumatoid Arthritis.
J. Med. Chem. 2015, 58 (1), 512–516.
Barbosa, A.; Blomgren, P. A.; Currie, K. S.; Krishna-
moorthy, R.; Kropf, J. E.; Lee, S. H.; Mitchell, S. A.;
Ortwine, D. F.; Schmitt, A. C.; Wang, X.; Xu, J.;
Young, W.; Zhang, H.; Zhao, Z.; Zhichkin, P. E. Pyri-
done and Aza-Pyridone Compounds and Methods of
Use. World Intellectual Property Organization WO
2011/140488 A1 November 10, 2011, pp 1–511.
Wuitschik, G.; Carreira, E. M.; Wagner, B.; Fischer,
H.; Parrilla, I.; Schuler, F.; Rogers-Evans, M.; Müller,
K. Oxetanes in Drug Discovery: Structural and Syn-
thetic Insights. J. Med. Chem. 2010, 53 (8), 3227–3246.
Liu, L.; Di Paolo, J.; Barbosa, J.; Rong, H.; Reif, K.;
Wong, H. Antiarthritis Effect of a Novel Bruton's Ty-
rosine Kinase (BTK) Inhibitor in Rat Collagen-
Induced Arthritis and Mechanism-Based Pharmaco-
kinetic/Pharmacodynamic Modeling: Relationships
Between Inhibition of BTK Phosphorylation and Effi-
cacy. J. Pharmacol. Exp. Ther. 2011, 338 (1), 154–163.
Katewa, A.; Wang, Y.; Hackney, J. A.; Huang, T.; Suto,
E.; Ramamoorthi, N.; Austin, C.; Bremer, M. C.; Chen,
J.; Crawford, J. J.; Currie, K. S.; Blomgren, P.; DeVoss,
J.; DiPaolo, J. A.; Hau, J.; Johnson, A.; Lee, W. P.;
Lesch, J.; DeForge, L.; Lin, Z.; Liimatta, M.; Lubach, J.;
McVay, S.; Modrusan, Z.; Nguyen, A.; Wang, J.; Liu,
L.; Wong, H.; Young, W. B.; Townsend, M. J.; Reif, K.
Specific Inhibition of Btk Blocks Pathogenic Plasma
Cell Signatures and Myeloid Cell Associated Kidney
Damage in IFNα-Driven Lupus Nephritis. J. Clin. In-
vest. Insight. 2017, 2(7), e90111.
(10)
Young, W. B.; Barbosa, J.; Blomgren, P.; Bremer, M.
C.; Crawford, J. J.; Dambach, D.; Gallion, S.; Hy-
mowitz, S. G.; Kropf, J. E.; Lee, S. H.; Liu, L.; Lubach,
J. W.; Macaluso, J.; Maciejewski, P.; Maurer, B.;
Mitchell, S. A.; Ortwine, D. F.; Di Paolo, J.; Reif, K.;
Scheerens, H.; Schmitt, A.; Sowell, C. G.; Wang, X.;
Wong, H.; Xiong, J.-M.; Xu, J.; Zhao, Z.; Currie, K. S.
Potent and Selective Bruton's Tyrosine Kinase Inhibi-
tors: Discovery of GDC-0834. Bioorg. Med. Chem. Lett.
2015, 25 (6), 1333–1337.
Johnson, A. R.; Kohli, P. B.; Katewa, A.; Gogol, E.;
Belmont, L. D.; Choy, R.; Penuel, E.; Burton, L.;
Eigenbrot, C.; Yu, C.; Ortwine, D. F.; Bowman, K.;
Franke, Y.; Tam, C.; Estevez, A.; Mortara, K.; Wu, J.;
Li, H.; Lin, M.; Bergeron, P.; Crawford, J. J.; Young, W.
B. Battling Btk Mutants with Noncovalent Inhibitors
That Overcome Cys481 and Thr474 Mutations. ACS
Chem. Biol. 2016, 11 (10), 2897–2907.
Watterson, S. H.; De Lucca, G. V.; Shi, Q.; Langevine,
C. M.; Liu, Q.; Batt, D. G.; Beaudoin Bertrand, M.;
Gong, H.; Dai, J.; Yip, S.; Li, P.; Sun, D.; Wu, D.-R.;
Wang, C.; Zhang, Y.; Traeger, S. C.; Pattoli, M. A.;
Skala, S.; Cheng, L.; Obermeier, M. T.; Vickery, R.;
Discenza, L. N.; D'Arienzo, C. J.; Zhang, Y.; Heimrich,
E.; Gillooly, K. M.; Taylor, T. L.; Pulicicchio, C.; McIn-
tyre, K. W.; Galella, M. A.; Tebben, A. J.; Muckelbau-
er, J. K.; Chang, C.; Rampulla, R.; Mathur, A.; Salter-
Cid, L.; Barrish, J. C.; Carter, P. H.; Fura, A.; Burke, J.
R.; Tino, J. A. Discovery of 6-Fluoro-5-(R)-(3-(S)-(8-
Fluoro-1-Methyl-2,4-Dioxo-1,2-Dihydroquinazolin-
3(4H)-Yl)-2-Methylphenyl)-2-(S)-(2-Hydroxypropan-
2-Yl)-2,3,4,9-Tetrahydro-1H-Carbazole-8-
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(19)
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13
14
15
16
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18
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21
22
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27
28
29
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33
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35
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37
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39
40
41
42
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45
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47
48
49
50
51
52
53
54
55
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57
58
59
60
(11)
(20)
(12)
(21)
(22)
(23)
Carboxamide (BMS-986142): a Reversible Inhibitor of
Bruton's Tyrosine Kinase (BTK) Conformationally
Constrained by Two Locked Atropisomers. J. Med.
Chem. 2016, 59 (19), 9173–9200.
(13)
Liu, J.; Guiadeen, D.; Krikorian, A.; Gao, X.; Wang, J.;
Boga, S. B.; Alhassan, A.-B.; Yu, Y.; Vaccaro, H.; Liu,
S.; Yang, C.; Wu, H.; Cooper, A.; de Man, J.; Kaptein,
A.; Maloney, K.; Hornak, V.; Gao, Y.-D.; Fischmann,
T. O.; Raaijmakers, H.; Vu-Pham, D.; Presland, J.;
Mansueto, M.; Xu, Z.; Leccese, E.; Zhang-Hoover, J.;
Knemeyer, I.; Garlisi, C. G.; Bays, N.; Stivers, P.; Bran-
dish, P. E.; Hicks, A.; Kim, R.; Kozlowski, J. A. Discov-
ery of 8-Amino-Imidazo[1,5- a]Pyrazines as Reversible
BTK Inhibitors for the Treatment of Rheumatoid Ar-
thritis. ACS Med. Chem. Lett. 2016, 7 (2), 198–203.
De Lucca, G. V.; Shi, Q.; Liu, Q.; Batt, D. G.; Beaudoin
Bertrand, M.; Rampulla, R.; Mathur, A.; Discenza, L.;
D'Arienzo, C.; Dai, J.; Obermeier, M.; Vickery, R.;
Zhang, Y.; Yang, Z.; Marathe, P.; Tebben, A. J.;
Muckelbauer, J. K.; Chang, C. J.; Zhang, H.; Gillooly,
K.; Taylor, T.; Pattoli, M. A.; Skala, S.; Kukral, D. W.;
McIntyre, K. W.; Salter-Cid, L.; Fura, A.; Burke, J. R.;
Barrish, J. C.; Carter, P. H.; Tino, J. A. Small Molecule
Reversible Inhibitors of Bruton's Tyrosine Kinase
(BTK): Structure-Activity Relationships Leading to
the Identification of 7-(2-Hydroxypropan-2-Yl)-4-[2-
Methyl-3-(4-Oxo-3,4-Dihydroquinazolin-3-
(24)
(14)
https://doi.org/10.1172/jci.insight.90111.
Yl)Phenyl]-9H-Carbazole-1-Carboxamide (BMS-
935177). J. Med. Chem. 2016, 59 (17), 7915–7935.
Gayko, U.; Fung, M.; Clow, F.; Sun, S.; Faust, E.; Price,
S.; James, D.; Doyle, M.; Bari, S.; Zhuang, S. H. Devel-
opment of the Bruton's Tyrosine Kinase Inhibitor Ib-
rutinib for B Cell Malignancies. Ann. N. Y. Acad. Sci.
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Discovery of Potent and Selective Tricyclic Inhibitors of Bruton’s Tyro-
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Xiaojing Wang, James Barbosa, Peter Blomgren, Meire C. Bremer, Jacob Chen, James J. Crawford, Wei Deng, Liming
Dong, Charles Eigenbrot, Steve Gallion, Jonathon Hau, Huiyong Hu, Adam R. Johnson, Arna Katewa, Jeffrey E.
Kropf, Seung H. Lee, Lichuan Liu, Joseph W. Lubach, Jen Macaluso, Pat Maciejewski, Scott A. Mitchell, Daniel F.
Ortwine, Julie DiPaolo, Karin Reif, Heleen Scheerens, Aaron Schmitt, Harvey Wong, Jin-Ming Xiong, Jianjun Xu,
Zhongdong Zhao, Fusheng Zhou, Kevin S. Currie, and Wendy B. Young*
For Table of Contents Use Only
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