Discovery of novel BTK inhibitors with carboxylic acids

Article abstract of DOI:10.1016/j.bmcl.2016.11.079

We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.

Full text of DOI:10.1016/j.bmcl.2016.11.079

Accepted Manuscript
Discovery of novel BTK inhibitors with carboxylic acids
Xiaolei Gao, James Wang, Jian Liu, Deodial Guiadeen, Arto Krikorian, Sobhana
Babu Boga, Abdul-Basit Alhassan, Oleg Selyutin, Wensheng Yu, Younong Yu,
Rajan Anand, Shilan Liu, Chundao Yang, Hao Wu, Jiaqiang Cai, Alan Cooper,
Hugh Zhu, Kevin Maloney, Ying-Duo Gao, Thierry O. Fischmann, Jeremy
Presland, My Mansueto, Zangwei Xu, Erica Leccese, Jie Zhang-Hoover, Ian
Knemeyer, Charles G. Garlisi, Nathan Bays, Peter Stivers, Philip E. Brandish,
Alexandra Hicks, Ronald Kim, Joeseph A. Kozlowski
PII:
S0960-894X(16)31244-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.079
BMCL 24474
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 November 2016
23 November 2016
24 November 2016
Please cite this article as: Gao, X., Wang, J., Liu, J., Guiadeen, D., Krikorian, A., Babu Boga, S., Alhassan, A-B.,
Selyutin, O., Yu, W., Yu, Y., Anand, R., Liu, S., Yang, C., Wu, H., Cai, J., Cooper, A., Zhu, H., Maloney, K., Gao,
Y-D., 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., Discovery of novel BTK inhibitors
with carboxylic acids, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.11.079
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Discovery of novel BTK inhibitors with
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Xiaolei Gao*, James Wang, Jian Liu*, Deodial Guiadeen, Arto Krikorian, Sobhana Babu Boga, Abdul-Basit Alhassan, Oleg Selyutin,
Wensheng Yu, Younong Yu, Rajan Anand, Shilan Liu, Chundao Yang, Hao Wu, Jiaqiang Cai, Alan Cooper, Hugh Zhu, Kevin
Maloney, Ying-Duo Gao, Thierry O. Fischmann, Jeremy Presland, My Mansueto, Zangwei Xu, Erica Leccese, Jie Zhang-Hoover, Ian
Knemeyer, Charles G. Garlisi, Nathan Bays, Peter Stivers, Philip E. Brandish, Alexandra Hicks, Ronald Kim, and Joeseph A. Kozlowski

Bioorganic & Medicinal Chemistry Letters
Discovery of novel BTK inhibitors with carboxylic acids
Xiaolei Gaoa,*, James Wanga, Jian Liua,* , Deodial Guiadeen a, Arto Krikorian a, Sobhana Babu Boga a, Abdul-Basit Alhassan a, Oleg
Selyutina, Wensheng Yua, Younong Yu a, Rajan Anand a, Shilan Liub, Chundao Yangb, Hao Wu b, Jiaqiang Caib, Alan Cooper a, Hugh Zhu a,
Kevin Maloneya, Ying-Duo Gaoa, Thierry O. Fischmann a, Jeremy Presland a, My Mansuetoa, Zangwei Xu a, Erica Leccese a, Jie Zhang-
Hoover a, Ian Knemeyer a, Charles G. Garlisi a, Nathan Bays a, Peter Stivers a, Philip E. Brandish a, Alexandra Hicks a, Ronald Kim a, and
Joeseph A. Kozlowski a
a Department of Early Development and Discovery Sciences, MRL, 126 East Lincoln Avenue, Rahway, NJ 07065 USA
b WuXi PharmaTech Co. Ltd, 288 FuTe Zhong Road, No. 1 Building, WaiGaoQiao Free Trade Zone, Shanghai 200131, P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We report the design and synthesis of a series of novel Bruton’s Tyrosine Kinase (BTK)
inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has
demonstrated much improved off-target selectivities including adenosine uptake (AdU)
inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4
based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and
human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK
inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat
and dog.
Keywords:
BTK inhibitor
Carboxylic acid ribose pocket
Adenosine uptake activity
Off target selectivities
hPBMC and hWB
2009 Elsevier Ltd. All rights reserved.
Bruton’s tyrosine kinase (BTK) is a Tec family kinase
expressed in immune cells including B cells, mast cells, and
macrophages.1,2 BTK plays a crucial role in B cell development,
proliferation, and signaling, and is also involved in cellular
signaling in myeloid cells.3 B cells have been suggested to
contribute to the pathology of autoimmune diseases such as
rheumatoid arthritis (RA), systemic lupus erythematous (SLE)
and multiple sclerosis,4 and pharmacological modulation of B
cell function with therapeutic antibodies such as rituximab and
belimumab have demonstrated clinical efficacy in autoimmune
diseases.5,6 Recently several groups reported the use of small
molecule BTK selective inhibitors as cancer therapies or for the
treatment of rheumatoid arthritis (RA).7 The covalent BTK
inhibitor Ibrutinib is currently approved for mantle cell
lymphoma and chronic lymphocytic leukemia.8 It is efficacious
in the rat collagen induced arthritis model (CIA), indicating its
potential use for the treatment of RA.8 Our group has recently
reported the discovery of potent and selective reversible non-
covalent BTK inhibitors based on 8-amino-imidazo[1,5-
a]pyrazines, as exemplified by compounds 1−3 (Figure 1).10
These inhibitors display high enzymatic potency, selectivity over
other kinases, favorable PK properties and are orally efficacious
in reducing paw thickness in a rat model of collagen induced
arthritis.
Figure 1. Structures of Ibrutinib and compounds 1-3.
However, while further profiling compound 3, we found that
it is a potent inhibitor of adenosine uptake (AdU) activity, which
caused concern due to the potential for unfavorable CV effects
since the cardiovascular actions of adenosine are well
documented.11 As a result, we undertook efforts to reduce AdU
activity while retaining the favorable overall profile of the series.
X-ray crystallography of the 8-amino-imidazo[1,5-a]pyrazines
bound to BTK show that the piperidine amide moiety occupies
the ribose binding pocket of the enzyme.10 Extensive SAR studies
were carried out to reduce the AdU activity and progress was
made through exploration of the ribose pocket SAR. We
discovered that replacing the amide with the carboxylic acid
moiety led to superior off-target selectivity. The initial lead
cyclohexylcarboxylic acid 4 (Figure 2) displayed significantly
improved AdU selectivity as well as improved hERG and Cav1.2
selectivities compared to the previous lead compound 3. Zhu et
al. has reported the inhibitory effect of a carboxylic acid group
on hERG binding and suggested that the negatively charged

carboxylate group causes unfavorable interaction within the
hERG channel binding cavity by electrostatic interaction.12
summarized in Table 1. Though several acid bioisosteres (13-15)
maintain improved AdU selectivity, they showed no
improvement in cellular potency; while others (16-20) showed
improved cellular potency but lost the superior AdU selectivity.
It is very likely that the improved AdU selectivities observed in
compound 13 to 15 are related to the low permeability of those
compounds, which also explains their poor cellular potency
observed in the hPBMC assay.
Table 1. Carboxylic acid bioisosteres Moiety in ribose pocket
Figure 2. Ribose pocket modification from piperidine amide to
cyclohexylcarboxylic acid
Preparation of this series of BTK inhibitors is depicted in
Scheme 1 and 2. (3-Chloropyrazin-2-yl)methanamine 5 is first
coupled with cis-3-(methoxycarbonyl)cyclohexane-1-carboxylic
acid
6
using HATU
as
coupling reagent and
BTK
IC50
(nM)
0.5
Cpd
#
hPBMC
AdU
(nM)
diisopropylethylamine as the base to provide compound 7 in 77%
yield, which underwent a cyclization to form the imidazole by
treatment with phosphorus oxychloride in acetonitrile to provide
intermediate 8. Crude 8 was utilized in the next step without
purification. Bromination of intermediate 8 was carried out using
N-bromosuccinimide in DMF at room temperature for 1 h,
producing intermediate 9 in 84% yield from compound 7. The
chloro intermediate 9 was converted to 10 in high yield with 2,4-
dimethoxybenzylamine in dioxane at room temperature for
overnight. LiOH hydrolysis followed by TFA deprotection
provided intermediate 11 in quantitative yield. SAR studies with
other carboxylic moieties in the ribose pocket were achieved with
various carboxylic acids esters to replace 6, which were either
commercially available or readily prepared. Bioisosteres of acid
are readily prepared from the ester 10.
Papp
(10-6 cm/s)
a
R
IC50a (nM)
98
7851
5600
7.0
2.0
4
13
1.2
0.5
1.4
0.3
0.6
0.3
0.5
0.2
648
14
15
16
17
18
1587
979
17
>10000
9086
168
150
155
44
3.0
NT
20
5
28
21
26
37
NT
NT
19
20
17
45
a The data are average of at least two repeated tests.
Scheme 1. Reagents. i. HATU, DIPEA, DMF, rt, 77%; ii. POCl3, CH3CN, 0
oC-rt; iii. NBS, DMF, rt, 84% for two steps; iv. 2,4-Dimethoxybenzylamine,
dioxanes, rt, 93%; v. 1N LiOH, THF/MeOH, 40 °C; vi. TFA, triethysilane, 70
°C, 99% for two steps.
We explored different ring sizes and projections of the carboxylic
acid in the ribose pocket with the representative examples shown
in Table 2. We discovered that all (21-25) demonstrated
enzymatic potency as well as good AdU selectivity; and some
displayed good cellular potency as well (24, 25). The 4-trans-
cyclohexyl carboxylic acid 21, and 3-cis-cyclopentyl carboxylic
acid 23, showed similar potency to compound 4, while the 4-cis-
cyclohexyl carboxylic acid 22 showed a decrease in cellular
potency. We also found that steric hindrance around the
carboxylic acid seemed to improve the hPBMC and hWB
potencies (compound 24 and 25). Compound 24 was later found
to have poor rat PK profiles, while compound 25 showed poor
off-target selectivity.
Boronic acid pinacol ester 12 was prepared as described in our
previous report.10 Suzuki coupling (Scheme 2) of 11 and 12,
using palladium catalyst such as PdCl2(dppf)CH2Cl2 and
potassium phosphate tribasic in dioxane /water (4/1) at 85°C for
6 h generated 48% of compound 4.
Scheme 2. Reagents. i. PdCl2(dppf)CH2Cl2, K3PO4, dioxane/water (4/1),
85°C, 6 h, 48%.
All BTK inhibitors were evaluated in a BTK enzymatic assay,
in a human peripheral blood mononuclear cell (hPBMC) and a
whole blood (hWB) functional assays.10 Compound 4 had
improved off target selectivities, but displayed weak hPBMC and
hWB potency, and demonstrated low bioavailability in the rat.
Initial optimization on 4 was carried out by exploring alternates
to the carboxylic acid. Representative examples (13-20) are

hPBMC
IC50
Table 2. Different unsubstituted carboxylic acids in ribose pocket
a
hWBa
(nM)
BTK IC50
(nM)
AdU
(nM)
a
Cpd
#
R1
R2
(nM)
0.2
0.3
5
99
308
274
26
27
hPBMC
IC50
(nM)
hWBa
(nM)
6
161
a
AdU
(nM)
a
BTK IC50
(nM)
Cpd#
R
28
29
0.2
0.4
9
264
459
0.4
1.2
98
2788
2307
7851
4
67
1909
6476
21
77
>10000
30
31
0.1
0.3
49
24
1267
67
812
22
23
0.5
0.8
1237
132
NT
NT
>10000
4435
9161
a The data are average of at least two repeated tests.
24
25
0.1
0.4
11
22
232
246
1927
7263
Unfortunately, the rat PK profile of compound 31 (Figure
3) was unacceptable, due to the very high clearance.
Metabolic identification studies suggested the major
metabolism was through glucuronidation of the carboxylic
acid. We therefore increased steric hindrance around the acid
moiety in an attempt to block or slow this pathway.
a The data are average of at least two repeated tests.
This result encouraged us to further explore analogs with
substituted ribose pocket ring. A series of bridgehead carboxylic
acids were prepared and tested (Table 3). Despite the excellent
hPBMC and hWB potencies, those bridgehead carboxylic acids
(26-28) displayed poor AdU selectivity. Compounds 29 and 30
showed reduced hWB potency, but interestingly, when we
introduced an ethoxy group onto the middle phenyl ring of
compound 26, compound 29, we observed potent enzyme,
hPBMC and hWB potency and reduced adenosine uptake
activity. SAR studies on the middle phenyl ring have suggested
that substitutions on middle phenyl rings have impact on AdU
activity.13
Figure 3. [2.2.2]-Bridgehead carboxylic acid 31
Several alpha-substituted carboxylic acids were then prepared
and tested (Table 4). Several of these analogs showed improved
hPBMC potency (compounds 32 and 35), though the hWB
potency still needed improvement. There is no apparent
correlation between hWB activity and protein binding (See the
unbound fraction data in Table 4 and Table 5). Compounds 33,
34 and 36 show weaker cellular potency.
Table 3. Bridgehead carboxylic acid moiety in ribose pocket
Table 4. Alpha-Substituted carboxylic acids moiety in ribose pocket

Table 6. Pharmacokinetic parameters and off target activities of compounds
hPBMC
IC50
(nM)
hWB
BTK
IC50
(nM)
37-40a
fub
AdU
(nM)
a
a
a
Cpd
#
R
(nM)
37
38
39
40
0.9
26
1071
2432
0.002
32
33
IV
PO
IV
PO
IV
PO
IV
13
5
PO
rat
Cl
9
3
2
2.8
2
(mL/min/kg)
3
128
62
NT
1938
5396
NT
NT
T1/2 (h)
F (%)
2.4
34
35
22
39
44
75
1.3
909
IV
PO
IV
PO
IV
PO
IV
13
PO
dog
1.1
4.5
27
604
NT
1711
2492
0.8
NT
Cl
(mL/min/kg)
T1/2 (h)
F (%)
5.8
546
36
77
a The data are average of at least two repeated tests;bUnbound fraction
in rat
hERG IC50
>60
54
>60
>60
(M)
We explored the impact of added steric bulk to the benzylic
position of the ribose pocket ring as well as adjacent to the
carboxylic acid. These analogs (37 to 40) displayed good
hPBMC and hWB potency while maintaining good AdU
selectivity. This series of compounds also had improved off-
target selectivity as compared to our previous lead compound 3,
and also displayed an excellent rat PK profile (Table 6).
Compound 40 demonstrated an acceptable dog PK profile.
Further profiling of compound 40 in in vivo studies is in progress.
Cav1.2 IC50
13
19
8
15
(M)
Nav1.5 IC50
>30
>30
>30
>30
(M)
a.
Formulations
for
both
IV
and
PO
are
both
DMSO/PEG400/Water:20/60/20
In summary, we described here a new carboxylic acid series
of reversible BTK inhibitors based on the 8-amino-imidazo [1,5-
a]pyrazine core. This series of compounds are characterized by
improved off-targets activities especially high AdU selectivity.
Analogs with steric bulk added near the carboxylate, i.e. in the
ribose pocket demonstrated potent BTK enzyme inhibition,
hPBMC, human whole blood activity, kinase selectivity, and
favorable pharmacokinetic profiles. Further investigation of the
SAR of this series of BTK inhibitors will be the subject of
another report.
Table 5. alpha-substituted carboxylic acids moiety in ribose pocket with a
tertiary methyl at benzylic position
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