Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes

Article abstract of DOI:10.1016/j.bmcl.2020.127390

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Full text of DOI:10.1016/j.bmcl.2020.127390

Journal Pre-proofs
Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-
a]pyrazine core featuring 3-position bicyclic ring substitutes
Jian Liu, Deodial Guiadeen, Arto Krikorian, Xiaolei Gao, James Wang,
Sobhana Babu Boga, Abdul-Basit Alhassan, Wensheng Yu, Oleg Selyutin,
Younong Yu, Rajan Anand, Jiayi Xu, Joseph Kelly, Joseph L. Duffy, Shilan
Liu, Chundao Yang, Hao Wu, Jiaqiang Cai, Chad Bennett, Kevin M.
Maloney, Sriram Tyagarajan, Ying-Duo Gao, Thierry O. Fischmann, Jeremy
Presland, My Mansueto, Zangwei Xu, Erica Leccese, Jie Zhang-Hoover, Ian
Knemeyer, Charles G. Garlisi, Peter Stivers, Philip E. Brandish, Alexandra
Hicks, Ronald Kim, Joseph A. Kozlowski
PII:
S0960-894X(20)30501-1
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127390
BMCL 127390
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
8 May 2020
1 July 2020
3 July 2020
Please cite this article as: Liu, J., Guiadeen, D., Krikorian, A., Gao, X., Wang, J., Babu Boga, S., Alhassan, A-B.,
Yu, W., Selyutin, O., Yu, Y., Anand, R., Xu, J., Kelly, J., Duffy, J.L., Liu, S., Yang, C., Wu, H., Cai, J., Bennett,
C., Maloney, K.M., Tyagarajan, S., Gao, Y-D., Fischmann, T.O., Presland, J., Mansueto, M., Xu, Z., Leccese, E.,
Zhang-Hoover, J., Knemeyer, I., Garlisi, C.G., Stivers, P., Brandish, P.E., Hicks, A., Kim, R., Kozlowski, J.A.,
Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position
bicyclic ring substitutes, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2
020.127390
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Potent, non-covalent reversible BTK
inhibitors with 8-amino-imidazo[1,5-
a]pyrazine core featuring 3-position bicyclic
ring substitutes
Leave this area blank for abstract info.
Jian Liu and et al.
CF3
CF3
N
N
O
N
O
N
NH
NH
NH2
Constraints
NH2
X
F
N
N
N
N
O
N
O
N
Y
3
: X = H, Y = O
4
: X = H, Y = CH2
5
: X = OMe, Y = CH2
2

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-
a]pyrazine core featuring 3-position bicyclic ring substitutes
a
a
a
a
a
a
Jian Liu *, Deodial Guiadeen , Arto Krikorian , Xiaolei Gao , James Wang , Sobhana Babu Boga , Abdul-
a
a
a
a
a
a
a
Basit Alhassan , Wensheng Yu , Oleg Selyutin , Younong Yu , Rajan Anand , Jiayi Xu , Joseph Kelly ,
Joseph L. Duffy , Shilan Liu , Chundao Yang , Hao Wu , Jiaqiang Cai , Chad Bennett , Kevin M.
Maloney , Sriram Tyagarajan , Ying-Duo Gao , Thierry O. Fischmann , Jeremy Presland , My Mansueto ,
Zangwei Xu , Erica Leccese , Jie Zhang-Hoover , Ian Knemeyer , Charles G. Garlisi , Peter Stivers , Philip
a
b
b
b
b
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
E. Brandish , Alexandra Hicks , Ronald Kim , Joseph A. Kozlowski
a
Department of Early Development and Discovery Sciences, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA
WuXi AppTec Co. Ltd, 288 FuTe Zhong Road, No. 1 Building, WaiGaoQiao Free Trade Zone, Shanghai 200131, PR China
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Bruton’s tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell
receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition,
and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for
the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on
8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with
inhibitors were also published, which provided great help for the SAR design. Here we report our
SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors
based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK
inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy
of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.
Keywords:
Bruton’s Tyrosine Kinase
Structure based drug design
Kinase selectivity
Rat CIA model
X-ray crystal structure
2
009 Elsevier Ltd. All rights reserved.
Bruton’s tyrosine kinase (BTK) is a Tec family kinase
expressed in certain immune cells including B cells, mast cells and
utilize C481 covalent binding for affinity, could still be efficacious
for patients with this mutation. Several research groups have
reported various non-covalent binding BTK inhibitors,
1
,2
11,12
macrophages. It plays a critical role in multiple pathways such
as the B cell receptor (BCR) and Fc receptor (FcR) signaling
cascades, where it regulates the survival, activation, proliferation,
differentiation and maturation of B cells.3,4 The role of BTK in
these pathways makes the enzyme a uniquely attractive target for
the treatment of B cell related diseases. BTK selective inhibitors
can be used as cancer therapies and for the treatment of rheumatoid
including our previously published BTK inhibitors based on 8-
O
N
O
N
O
NH
NH2
N
NH2
N
NH2
N
O
5
arthritis (RA). Currently the three approved BTK inhibitor drugs
N
N
N
N
6
7
8
N
O
HN
are ibrutinib, acalabrutinib, and zanubrutinib (Figure 1). Many
O
N
O
9
N
BTK inhibitors are in clinical trials. These approved drugs are
N
irreversible covalent binding inhibitors. The covalent binding
BTK inhibitor ibrutinib was approved for the treatment of mantle
cell lymphoma and chronic lymphocytic leukemia. However, a
small portion (5.3%) of the patients on ibrutinib therapy
encountered relapse for chronic lymphocytic leukemia. This
relapse was primarily caused by a cysteine to serine mutation at
C481, which is the covalent binding residue. Thus, the mutation
results in significant loss in binding affinity of ibrutinib to mutant
Ibrutinib
Acalabrutinib
Zanubrutinib
CF3
CF3
CF3
N
N
N
O
O
N
O
NH
NH
NH
NH2
NH2
NH2
Constraints
X
F
N
N
N
N
1
0
N
BTK. A potent non-covalent BTK inhibitor, which does not
N
N
N
O
O
O
N
O
N
N
Y
3
4
5
: X = H, Y = O
1
2
: X = H, Y = CH2
Cyclization
: X = OMe, Y = CH2
Figure 1

amino-imidazol[1,5-a]pyrazine core, exemplified by compound 1
converted to (6R,8aS)-6-(8-chloroimidazo[1,5-a]pyrazin-3-yl)
hexahydro-3H-oxazolo[3,4-a]pyridin-3-one 12 after being treated
with phosphoryl trichloride in acetonitrile with a quantitative
yield. Bromation of 12 using NBS in DMF provided an excellent
yield of bromo compound 13. The 8-chloro substituent in
compound 13 was then converted to 8-amino by treatment with
and 2 (Figure 1).1
3,14,15
Ibrutinib showed excellent efficacy in the
rat collagen induced arthritis model (CIA), indicating its potential
use for the treatment of RA.16 Our previously reported non-
covalent BTK inhibitor 2 also demonstrated dose-dependent
efficacy in the rat CIA model.
ammonium hydroxide in isopropanol in a sealed reaction vessel at
We also reported the X-ray crystal structure of compound 2 in
o
1
00 C for 7 hours to give a moderate yield of 14. Suzuki coupling
1
3
the binding pocket of BTK. The cyclopropylamide substituent on
the 3-piperidine has a trans conformation forced by the methyl
group with the carbonyl oxygen forming a hydrogen bond with the
C481 NH. The alpha carbon of the amide is in the position that can
be cyclized to the piperidine 2-carbon to fix the amide carbonyl in
the proper orientation for the hydrogen binding with C481
backbone NH. Thus, we designed compounds 3 (Y = O) and 4 (Y
1
3
of 14 with boronic ester 15 afforded the final compound 3.
O
O
O
O
HO
iii, iv
CO2Me
O
ii
O
O
i
N
N
CO2Me
N
N
H
O
6
16
17
18
CF3
CF3
O
=
2
CH ) with a cyclic carbamate or lactam ring constraint. We
O
modeled that the ring constraints would fix the carbonyl oxygen in
the direction to hydrogen bonding with C481 backbone NH, which
potentially could increase the binding potency while also
improving the PK profile.
NH2
O
N
Br
B
N
H
N
N
O
NH
N
N
15
N
v
NH2
N
O
N
4
N
19
O
O
O
O
N
O
O
HO
i, ii
O
iii, iv, v
vi
O
N
N
N
N
Cbz OH
OAc
O
6
7
8
9
O
o
Scheme 2. i. Ethyl 2-oxoacetate, Ac
2 2
O, 130 C, 48 h, 73%; ii. Pd(OH) /C,
Cl
Br
AcOH, H (40 psi), 18 h; iii. TEA, MeOH, rt, 18 h; iv. NaOMe (2 eq), MeOH,
50 C, HCl, 76% three steps; v. Pd(dppf)Cl , K CO , dioxane, 100 C, 2 h, 36%.
2 2 3
2
N
Cl
O
N
N
N
O
N
o
o
NH2
viii
N
ix
N
N
N
O
1
0
Cl
Cl
NH
N
O
N
vii
N
N
O
O
13
11
12
O
We then synthesized the bicyclic lactam acid 18, which could
be converted to the final target 4. The preparation of 4 (X = CH
CF3
O
CF3
2
)
O
N
with an alternative lactam ring constraint started with the same
starting material methyl 6-methylnicotinate 6 (Scheme 2). It was
heated together with ethyl 2-oxoacetate in acetic anhydride at 130
NH2
O
Br
B
N
H
N
NH
N
O
N
15
x
N
NH2
xi
o
N
O
N
C for two days to afford 16 with good yield. Hydrogenation was
N
N
3
O
14
carried out in acetic acid catalyzed by palladium hydroxide on
carbon under 40 psi hydrogen atmosphere overnight to generate
the fully reduced piperidine compound 17. The lactam was formed
as a cis/trans mixture when 17 was stirred in methanol along with
triethylamine at room temperature for 18 hours. The lactam was
N
O
O
o
Scheme 1. i. mCPBA, CH
NaBH
two steps; v. HCl, MeOH, reflux, 24 h, 55%; vi. LiOH, THF/H
2
Cl
2
, rt; ii. Ac
2
O, 130 C, 1h, 35% two steps; iii.
o
3
CN, AcOH, rt; 24 h; iv. Cbz-Cl (2eq), NaHCO
3
, 0 C to rt, 24 h, 34%
O, rt, 24 h,
, CH CN, rt, 15
3
/ PrOH, 100 C, 7 h, sealed tube,
o
then treated with sodium methoxide in methanol at 50 C to
2
i
hydrolyze the ester to acid, and at the same time the cis/trans
mixture of acid isomerized to the thermodynamically more stable
trans acid 18. The acid 18 was precipitated as a racemate in water
as white solid when treated with hydrochloric acid. Following the
same chemistry described in Scheme 1 for the preparation of 14,
acid 18 was converted to the intermediate 19 as a racemate which
was resolved to two enantiomers. The desired (6R,8aS)
enantiomer 19 was coupled with 15 to form the final product 4 by
1
00%; vii. PrOCOCl (1eq), TEA (2eq), 3 h, 73%; viii. POCl
h, 94%; ix. NBS, DMF, rt, 1 h, 83%; x. NH
4%; xi. Pd(dppf)Cl , K CO , dioxane, 100 C, 2 h, 36%.
2 2 3
3
3
i
o
o
7
The preparation of 3 is depicted in Scheme 1, with all reaction
reagents and conditions noted.17 The synthesis started from methyl
-methylnicotinate 6, which was treated with mCPBA for the
6
formation of pyridine N-oxide. The N-oxide was then reacted with
acetic anhydride to the N-oxide acetate, followed by a
rearrangement reaction to produce methyl 6-(acetoxymethyl)
nicotinate 7. The pyridine ring in 7 was then reduced to the
piperidine by sodium cyano borohydride. The formed piperidine
nitrogen was protected by reacting with benzyl chloroformate.
Later the acetate was hydrolyzed by lithium hydroxide in
THF/water to provide trans (+/-) 1-benzyl 3-methyl-6-
17
a Suzuki coupling reaction.
Table 1. In vitro BTK inhibition data for 2, 3 and 4
Compound
BTK Enzymatic hPBMC
Human whole
Blood
a
a
a
Assay
Assay
IC50 (nM)
IC50 (nM)
IC50 (nM)
2
3
4
0.31
0.083
0.1
4.0
5.8
2.5
94
36
24
(
hydroxymethyl)piperidine-1,3-dicarboxylate 8. The racemate 8
was resolved by chiral HPLC to provide 1-benzyl 3-methyl
3S,6R)-6-(hydroxymethyl)piperidine-1,3-dicarboxylate 8. The
(
a.
Reported values are the average of > 2 independent measurements
with standard deviation less than 3-fold of the reported mean
cyclic carbamate was formed along with the hydrolysis of the
methyl ester when 8 was treated with lithium hydroxide in mixed
solvent of tetrahydrofuran and water at room temperature
overnight to generate compound 9 (6S,8aR)-3-oxohexahydro-3H-
oxazolo[3,4-a]pyridine-6-carboxylic acid. Acid 9 was then
coupled with (3-chloropyrazin-2-yl)methanamine 10 after being
activated by isopropyl chloroformate to form (6S,8aR)-N-((3-
chloropyrazin-2-yl)methyl)-3-oxohexahydro-3H-oxazolo[3,4-
a]pyridine-6-carboxamide 11. The amide 11 was subsequently

O
O
O
Along with the known inhibitor 2, BTK inhibitors 3 and 4 were
evaluated in the BTK enzymatic binding assay, the hPBMC B cell
functional assay and the human whole blood B cell functional
assay. The data are summarized in Table 1. Compared to 2, both
the cyclized 3 and 4 with carbamate and lactam as the bicyclic
constraints showed improved binding potency to BTK enzyme by
more than 3-fold. Furthermore, the cellular and whole blood
O
i, ii
O
iii
HO
N
N
N
Cbz OH
Cbz OH
O
1
3
O
8
20
21
iv, v
O
O
HO
vi
O
CF3
N
N
23
O
N
Cbz OH
O
O
NH
CF3
O
22
N
NH2
NH
N
N
N
NH2
N
25
N
O
N
N
O
N
O
24A and 24B
O
compound 24A, 24B and 25, applying the similar chemistry in
1
7
Scheme 1.
o
Scheme 3. i. Dess-Martin, CH
0%; iii. LiOH (2eq), THF/H O, rt, 12 h, 75%; iv. Dess-Martin, CH
0%; v. MeMgBr, THF, -78 to -25 C, 4 h, 33%; vi. LiOH (2eq), THF/H
2
Cl
2
, rt, 85%; ii. MeMgBr, THF, -78 C,
Cl , rt,
O, 3
5
6
2
2
2
o
2
h, 79%.
F3C
N
O
NH
Table 2. SAR data of 3-position bicyclic substitutes
NH2
X
N
activities were also significantly improved. The enantiomer of
N
N
compound 4 is 150-fold less potent.
R
Compound #
R, X
Enzymatic
Assay
IC50 (nM)
hPBMC
Assay
IC50 (nM)
Human
whole Blood
EC50 (nM)
a
a
Figure 2. Overlay of X-ray crystal structures of compound 2 (green) and 3
purple) binding to BTK enzymes.
(
N
O
We were able to get the X-ray co-crystal structure of 3 bound
0.13
4.9
86
63
58
O
to the BTK enzyme. Figure 2 shows the overlay of the crystal
structures of both 2 and 3 binding to the BTK enzyme. The
carbamate derivative 3 has the similar binding mode to 2 in the
back pocket, and the hinge region as described in the previous
2
2
4A
4B
X = H
X =H
N
O
0
0
.11
.12
3.2
2.8
O
1
3
publication. These interactions provide the high binding potency
and the selectivity. The carbonyl of the carbamate in the bicyclic
ring in 3 (purple) is locked in the conformation to form a hydrogen
bond with the amide NH of C481. In the meantime, the carbamate
oxygen forms a hydrogen bond with the side chain amide of N484,
which provides the extra binding potency for 3 compared to 2.
N
O
O
2
5
X = H
O
O
N
O
0.26
25
7.2
263
NT
2
6
X = H
The carbamate functional group is potentially a soft spot for
metabolism, specially at the carbon connected to the carbamate
oxygen. To block the potential metabolism, we prepared
compounds with methyl or gem-dimethyl substitution (Scheme 3).
The alcohol 8 was oxidized to the aldehyde using Dess-Martin
reagent in methylene chloride at room temperature, followed by
N
O
582
O
27
X = H
N
O
7
.2
403
NT
N
2
2
3
8
9
0
X = H
X = H
X = F
o
addition of the methyl magnesium bromide in THF at -78 C to
N
0.67
1.7
39
39
204
NT
78
provide the alcohol compound 20 in a moderate yield (50%).
Cyclization occurred when 20 was stirred under a basic condition
in the mixture of tetrahydrofuran/water and lithium hydroxide to
form the cyclic carbamate 21 with a good yield as a mixture of two
diastereomers. In the same sequence, the secondary alcohol 20
could be oxidized again by Dess-Martin reagent to a ketone,
followed by addition of the methyl magnesium bromide to provide
the tertiary alcohol 22. When 22 was treated with lithium
hydroxide in tetrahydrofuran/water, the cyclic carbamate bicyclic
carboxylic acid 23 with the gem-dimethyl formed in a good yield
O
N
O
N
0.1
5.8
O
3
1
X =F
X =H
O
N
0
.26
16
104
N
(
79%). Bicyclic acids 21 and 23 were converted to the final
32

a.
Reported values are the average of > 2 independent measurements with
correlating with the increased size of substitutes. Compound 5
showed better selectivity than 4, while 37 had better selectivity than
standard deviation less than 3-fold of the reported mean
NT = Not Tested
5
.
CF3
N
O
To explore other constrained bicyclic substitutes on the 3-
NH
Table 3: SAR data for phenyl 3-substitutes
position of the core imidazo[1,5-a]pyrazine, a series of bicyclic
analogs were prepared (Table 2). The precursor bicyclic acids
were synthesized and then the same chemistry in Scheme 1 was
applied to generate the final target molecules in the table.17 This
series of compounds with 8-amino-imidazo[1,5-a]pyrazine core
and various selected 3-position bicyclic ring substitutes were
evaluated in the BTK enzymatic, cellular and whole blood assays.
The BTK enzyme tolerated many changes with these bicyclic ring
substitutes on the 3-position. Compound 24a, 24b and 25 with
methyl substitutes on the carbon alpha to the carbamate oxygen
were tolerated with similar activity to 3 in enzymatic, cellular and
whole blood assays. This series of compounds maintained most of
the BTK inhibition potency for compounds 26 with morpholinyl
bicyclic lactam, 29 with octohydroindolinzinyl, 30 with
octohydro-4H-quinolizinonyl, 31 with hexahydropyrido[2,1-
c][1,4]oxazin-4(3H)-onyl, and 32 with 2-methyloctahydro-4H-
pyrido[1,2-a]pyrazin-4-onyl. However, compound 27 with the
bicyclic morpholine carbamate and 28 with the bicyclic methyl
urea are much less potent, which may have been caused by
unfavored interactions with the BTK enzyme.
NH2
X
N
N
N
N
O
Compound #
X
BTK Enzymatic hPBMC Assaya
Human whole
Blood EC50 (nM)
a
Assay IC (nM)
5
0
IC50 (nM)
4
5
3
H
0.1
2.5
24
OMe
F
0.21
0.11
0.16
0.48
0.58
0.58
5.7
17
30
3
4.0
101
92
34 OEt
OCF
10
3
5
2
H
20
415
NT
494
NT
NT
NT
NT
NT
NT
NT
NT
36 OH
11
c
3
7
O Pr
12
i
38
O Pr
41
3
9
OPh
CONH
COOH
CN
11
310
>3000
1410
267
822
>3000
74
Previously, we demonstrated the SAR of substitutes on the
middle phenyl that variable substitution X at the 3-position is
40
2
348
83
4
1
tolerated by BTK enzyme for other reported series of BTK
inhibitors based on 8-amino-imidazo[1,5-a]pyrazine core.13,14,15
42
6.3
Thus we further explored this SAR on the impact of BTK
inhibition activity with different substitutes on this position based
on compound 4. These compounds can be prepared by applying
boronic esters like 15 in Scheme 1 with different substitutes on the
phenyl ring 3-position.17 The compounds were evaluated in the
BTK enzymatic, cellular and whole blood assays and the data are
shown in Table 3. There is limited space around the middle phenyl
4
4
4
3
4
5
CF
CF
2
CH
3
831
>1000
10
3
OCF
3
b. Reported values are the average of >2 independent measurements with
standard deviation less than 3-fold of the reported mean
NT = Not Tested
to allow only small substitutes (X) on the phenyl 3-position, such
c
Table 4: Selectivity of compound 3, 4, 5, 25 and 37 over Tec and Src
as OMe (5), F (33), OEt (34), OCF
2
H (35), OH (36), and O Pr
family kinases
(37), all of which maintain the BTK enzymatic inhibition with sub-
nanomolar potency. However, there is significantly 20-fold loss of
potency when the substitution was changed from cyclopropyl to a
slightly bigger isopropyl (38). The bigger size substitution like
phenoxy (39) resulted in further loss of potency. The polar and
electron-withdrawing substitutes exemplified by carbamide (40),
Kinase
Comp 3
Comp 4
Comp 5 Comp 25
Comp 37
IC50 (nM)
(fold x)
IC50 (nM) IC50 (nM)
IC50 (nM) IC50 (nM)
(fold x)
(fold x)
0.1
(fold x)
(fold x)
BTK
BLK
BMX
CSK
0.083
0.21
0.12
0.58
309 (532)
64 (110)
carboxylic acid (41), cyano (42), CF
45) all caused significant loss of the activity in enzymatic and
cellular assays.
2 3 3 3
CH (43), CF (44) and OCF
22 (265)
13 (157)
8.3 (83)
11 (110)
101 (480)
19 (90)
17 (140)
5.6 (47)
(
290
162
227
350
717 (1236)
Most of the potent BTK inhibitors were screened through a
small kinome panel with representative kinases from the Tec and
Src family to evaluate the selectivity of this series BTK inhibitors
over other kinases. The data for compound 3, 4, 5, 25 and 37 are
listed in Table 5 with the IC50 and fold of selectivity over BTK
enzyme. All these BTK inhibitors demonstrated high selectivities
over other Tec and Src family kinases. Comparing the
cyclocarbamate 3 to the lactam 4, compound 3 has better selectivity
(3500)
(1620)
(1080)
(2900)
ERBB4
FGR
34 (410)
26 (313)
46 (554)
18 (219)
24 (240) 160 ((762)
23 (190)
34 (283)
36 (300)
3300 (5700)
263 (453)
530 (913)
NT
34 (340)
87 (870)
64 (304)
FRK
154 (733)
FYN
1230
(5900)
212 7400 (12758)
(1700)
(
fold) across most kinases in the panel. This improved selectivity
LCK
11 (132)
49 (590)
50 (600)
92 (1110)
14 (169)
92 (1110)
5.9 (59)
20 (200)
42 (420)
33 (330)
9.1 (91)
63 (630)
26 (124)
162 (771)
84 (400)
71 (338)
16 (76)
13 (110)
79 (660)
24 (200)
69 (570)
8.0 (67)
27 (220)
182 (313)
865 (1500)
320 (550)
946 (1630)
61(115)
can be rationalized by the hydrogen bond interaction of the
carbamate oxygen with N484 side chain amide described in the X-
ray crystal structure above. This interaction does not exist with the
lactam structure of 4. When the gem-dimethyl was introduced on
the carbon next to the oxygen in the carbamate, compound 25
showed lower selectivities compared to 3. The methoxy and
cyclopropoxy substitutes on the phenyl 3-position have significant
impact on the BTK selectivity, with an increased selectivity
LYNB
PTK6
SRC
SRMS
TEC
67 (319)
277 (477)

To understand the excellent selectivities of compound 5 and 37,
we obtained the X-ray crystal structures for 5 (purple) and 37
green) bound to BTK enzyme, with overlay shown in Figure 3.
TXK
46 (554)
35 (420)
33 (330)
12 (120)
102 (485)
54 (257)
15 (125)
30 (250)
312 (537)
430 (740)
1
8
YES1
(
The data are average of at least two repeated tests. NT: not tested.
The methoxy substitution on 5 and the cyclopropoxy on 37 occupy
different pockets of the BTK enzyme. Looking from the bicyclic
ring toward the binding pocket, the methoxy on 5 is pointing to the
direction of the gate keeper T474 surrounded by a tight
hydrophobic pocket, while the cyclopropoxy on 37 is fitting in the
opposite side in a relatively bigger pocket. These two pockets of
the BTK enzyme allow the substitutions on the phenyl 3-position
to provide improved selectivity of BTK over the other kinases
tested. But the limited size of these two pockets cannot fit
substitutes with size bigger than cyclopropyl.
Several BTK inhibitors of this series were then evaluated in
pharmacokinetic tests in preclinical species rats and dogs. The
pharmacokinetic parameters for compound 2, 3, 4, 5, 25 and 37 are
listed in Table 5. Compound 3 with the carbamate ring constraint
had similar clearance (Cl = 17 ml/min/kg) and half life time (t1/2
=
5.1 h) when dosed IV ( 2 mg/kg) compared to 2 (Cl = 18
1
3
ml/min/kg, t1/2 = 1.8 h). However, 3 had very low oral
bioavailability (F = 3.6%), possibly due to the high first pass
clearance in liver.
Figure 3. Overlay of the X-ray crystal structures for 5 (purple) and 37 (green),
view from the 3-position of the bicyclic ring.
Table 5. Pharmacokinetic parameters of compound 2, 3, 4, 5, 25 and 37
Compound
Rata
2
3
4
5
25
37
IV
dose
2
PO
dose
5
IV
dose
2
PO
dose
5
IV
dose
2
8.0
0.78
7.8
PO
dose
5
IV
dose
2
PO
dose
5
IV
dose
2
PO
dose
5
IV
dose
2
PO
dose
5
Dose (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
18
17
53
26
42
2.6
3.1
1.8
1.2
5.1
5.1
4.0
1.1
3.0
5.6
2.3
3.6
4.2
1.4
2.3
AUCN (M*h)
T
C
T
1/2 (h)
max (M)
max (h)
2.8
0.40
1.0
22
0.34
0.25
3.6
1.3
0.75
46
0.05
2.1
9.5
1.0
2.5
160
0.14
3.5
14
F (%)
Dogb
IV
dose
1
1.5
0.65
20
PO
dose
2
IV
dose
PO
dose
IV
dose
1
PO
dose
2
IV
dose
1
PO
dose
2
IV
dose
PO
dose
IV
dose
PO
dose
Dose (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
NT
NT
11
20
NT
NT
NT
NT
1.5
2.8
3.6
3.0
1.6
3.4
AUCN (M*h)
T
C
T
1/2 (h)
max (M)
max (h)
6.5
3.9
2.0
82
1.7
1.0
100
1.1
1.5
108
F (%)
a. Rats for PK test are Wistar Han
b. Dogs for PK test are beagles
To address the high clearance of compound 3, the gem-
dimethyl was introduced on the carbon connected to the carbamate
oxygen in compound 25. A much-improved oral bioavailability (F
which lead to low oral exposure. Compound 5 had high clearance
and volume distribution in dogs, however, comparable oral
exposure in dogs to 4.
=
160%) in rats was observed, although the total clearance was still
high. The first path clearance by liver was reduced significant for
5. The lactam constrained compound 4 showed excellent PK
Based on the overall profile of the lead BTK inhibitors listed
in Table 5, including potency in enzymatic, cellular and whole
blood assays, the kinase selectivities and the PK data, we chose to
evaluate compounds 4 and 5 in the rat model of collagen induced
arthritis (CIA) for their in vivo efficacy. In a prophylactic
treatment protocol, compound 4 demonstrated a dose-dependent
2
profile in both rats and dogs with low clearance, long half-lives
and high oral bioavailability. However, when alkoxy substitutions
were installed for 5 (MeO) and 37 ( PrO) on the middle phenyl
c
13
ring, the clearances for both compounds in rats are much higher

(
1, 3, 10, 30 mg/Kg, PO, QD) decrease in paw thickness, as
2.
Tsukada, S.; Saffran, D. C.; Rawlings, D. J.; Parolini, O.; Allen, R. C.;
Klisak, I.; Sparkes, R. S.; Kubagawa, H.; Mohandas, T.; Quan, S.;
Belmont, J. W.; Cooper, M. D.; Conley, M. E.; Witte, O. N. 1993, 72,
indicated by significant reduction (p<0.0001) of paw thickness
through day 30 consequent to CIA induction (Figure 4A). Due to
the less optimal PK profile of compound 5 in rats, higher doses
2
79-290.
3
.
Kurosaki, T.; Hikida, M. Haslam, Imm. Rev. 2009, 228, 132-48.
Cunningham-Rundles, C.; Ponda, P. P. Nat. Rev. Immunol. 2005, 5, 880-
892.
For review: Norman, P. Investigational Bruton’s tyrosine kinase
inhibitors for the treatment of rheumatoid arthritis Exp. Opin. Inv. Drugs.
(15, 30, 60 and 120 mg/Kg, PO, QD) were required to achieve the
4.
similar efficacy in this CIA model with a dose-dependent decrease
in the paw thickness (Figure 4B). We were able to demonstrate the
in vivo efficacy in the preclinical species with this series of non-
covalent reversible BTK inhibitors.
5
6
7
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2
019, 25(8), 891-899,.
Charalambous, A.; Schwarzbich, M. A.; Witzens-Harig, M. Ibrutinib. In:
Martens U. (eds) Small Molecules in Hematology. Recent Results in
Cancer Research, 2018, 212, 242. Springer, Cham.
Kriegsmann, K.; Kriegsmann, M.; Witzens-Harig, M. Acalabrutinib, A
Second-Generation Bruton’s Tyrosine Kinase Inhibitor. In: Martens U.
(eds) Small Molecules in Hematology. Recent Results in Cancer
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0. Woyach, J. A.; Furman, R. R.; Liu, T.-M.; Ozer, H. G.; Zapatka, M.;
Ruppert, A. S.; Xue, L.; Li, D. H.; Steggerda, S. M.; Versele, M.; Dave,
S. S.; Zhang, J.; Yilmaz, A. S.; Jaglowski, S. M.; Blum, K. A.; Lozanski,
A.; Lozanski, G.; James, D. F.; Barrientos, J. C.; Lichter, P.; Stilgenbauer,
S.; Buggy, J. J.; Chang, B. Y.; Johnson, A. J.; and Byrd, J. C. Resistance
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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.;
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L.; Pulicicchio, C.; McIntyre, K. W.; Galella, M. A.; Tebben, A. J.;
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Figure 4. Dose dependent changes in paw thickness in a rat CIA model at
different doses of 4 and 5. Pred = Prednisolone (postive control)
1
1
3. Liu, J.; Guiadeen, D.; Krikorian, K.; Gao, X.; Wang, J.; Boga, S. B.;
Alhassan, A.-B.; Yu, Y.; Vaccaro, H.; Liu, S.; Yang, C.; Wu, H.; Cooper,
A.; DeMan, J.; Kaptein, A.; Maloney, K.; Honak, V.; Gao, Y.-G.;
Fischmann, T. O.; Raaijmakers, H.; Presland, J.; Mansueto, M.; Xu , Z.;
Leccese, E.; Zhang-Hoover, J.; Knemeyer, I.; Garlisi, C. G.; Bays, N.;
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C.; Wu, H.; Cai, J.; Cooper, A.; Zhu, Hugh; Maloney, K.; Gao, Y.;
Fischmann, T. O.; Presland, J.; Mansueto, M.; Xu, Z.; Leccese, E.;
Zhang-Hoover, J.; Knemeyer, I.; Garlisi, C. G.; Bays, N.; Stivers, P.;
Brandish, P. E.; Hicks, A.; Kim, R.; Kozlowski, J. A. Bioorg. Med. Chem.
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H.; Desai, J.; Maloney, K.; Gao, Y-D.; Fischmann, T.O.; Presland, J.;
Mansueto, M.; Xu, Z.; Leccese, E.; Knemeyer, I.; Garlisi, C.G.; Bays, N.;
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In summary, we described the discovery of a series of non-
covalent reversible BTK inhibitors based on the 8-
aminoimidazo[1,5-a]pyrazine core with various 3-position
bicyclic ring substitutes. The cyclic ring constraints lock the
carbonyl in the direction to form hydrogen bond with C481
backbone amide NH, which is demonstrated in the X-ray crystal
structure of the inhibitor 3 bound to BTK. At the same time, we
observed an additional interaction of 3 with N484. Various
bicyclic ring substitutes were tolerable to fit into the BTK enzyme
binding pockets and maintained potency in the enzymatic, cellular
and whole blood assays. Substituents on the middle phenyl 3-
position significantly impacted the BTK binding potency and
selectivity against the Tec and Src family kinases. This selectivity
can be rationalized by the orientation of 3-substituents on 5 and 37
within the pockets around the phenyl ring in the X-ray crystal
structures. Compound 4 and 5 have combined desired properties,
with potent activities in the BTK enzymatic, cellular, human whole
blood assays, the kinase selectivity and in vivo pharmacokinetic
profile in preclinical species. These orally bioavailable non-
covalent reversible BTK inhibitors 4 and 5 displayed dose-
dependent efficacy in reducing the paw thickness in the rat
collagen induced arthritis model.
1
1
17. Detail synthetic experimental procedure can be found at: Kim, R. M.; Liu,
J.; Gao, X.; Boga, S. B.; Guiadeen, D.; Kozlowski, J. A.; Yu, W.; Anand,
R.; Yu, Y.; Selyutin, O. B.; Gao, Y.; Wu, H.; Liu, S.; Yang, C.; Wang,
H. “BTK inhibitors” U.S. Pat. Appl. Publ. 2014, US 20140206681 A1.
1
8. The data of X-ray crystal structures for BTK inhibitors 3, 5 and 37 is up-
loaded to the Protein Data Bank (www.pdb.org) with codes 6X3N, 6X3O
and 6X3P respectively.
References and notes
1
.
Vetrie, D.; Vorechovsky, I.; Sideras, P.; Holland, J.; Da-vies, A.; Flinter,
F.; Hammarstrom, L.; Kinnon, C.; Levinsky, R.; Bobrow, M.; Smith, C.
I. E.; Bentley, D. R. Nature 1993, 361, 226−233.