Discovery of 8-Amino-imidazo[1,5-a]pyrazines as Reversible BTK Inhibitors for the Treatment of Rheumatoid Arthritis

Article abstract of DOI:10.1021/acsmedchemlett.5b00463

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. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.

Full text of DOI:10.1021/acsmedchemlett.5b00463

Letter
pubs.acs.org/acsmedchemlett
Discovery of 8‑Amino-imidazo[1,5‑a]pyrazines as Reversible BTK
Inhibitors for the Treatment of Rheumatoid Arthritis
Jian Liu,*,† Deodial Guiadeen,† Arto Krikorian,† Xiaolei Gao,† James Wang,† Sobhana Babu Boga,†
Abdul-Basit Alhassan,† Younong Yu,† Henry Vaccaro,† Shilan Liu,‡ Chundao Yang,‡ Hao Wu,‡
Alan Cooper,† Jos de Man,† Allard Kaptein,† Kevin Maloney,† Viktor Hornak,† Ying-Duo Gao,†
Thierry O. Fischmann,† Hans Raaijmakers,† Diep Vu-Pham,† 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 Joseph A. Kozlowski†
†Department of Early Development and Discovery Sciences, Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, New
Jersey 07065, United States
‡WuXi PharmaTech Co. Ltd, 288 FuTe Zhong Road, No. 1 Building, WaiGaoQiao Free Trade Zone, Shanghai 200131, P. R. China
S
* Supporting Information
ABSTRACT: 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. We report a series of compounds based on 8-amino-
imidazo[1,5-a]pyrazine that are potent reversible BTK
inhibitors with excellent kinase selectivity. Selectivity is
achieved through specific interactions of the ligand with the
kinase hinge and driven by aminopyridine hydrogen bondings
with Ser538 and Asp539, and by hydrophobic interaction of
trifluoropyridine in the back pocket. These interactions are
evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds
show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.
KEYWORDS: BTK, rheumatoid arthritis (RA), rat CIA model, kinase selectivity,
human peripheral blood mononuclear cells (hPBMC), whole blood, pharmacokinetic, X-ray crystal structure
heumatoid arthritis (RA) is an autoimmune disease that
causes chronic inflammation of the joints, the surrounding
the treatment of B cell related diseases. In fact, several groups
have reported small molecule BTK selective inhibitors as cancer
therapies or for the treatment of RA,6 with ibrutinib currently
approved for mantle cell lymphoma and chronic lymphocytic
leukemia.7 Ibrutinib also shows excellent efficacy in the rat
collagen induced arthritis model (CIA), indicating its potential
use for the treatment of RA.8 Ibrutinib is an orally administered
selective BTK inhibitor, which covalently binds to the
sulfhydryl group of C481 leading to irreversible inhibition of
its kinase activity. A small portion (5.3%) of patients
encountered relapse during ibrutinib therapy for chronic
lymphocytic leukemia, majorly due to a cysteine to serine
mutation at C481, which results in a reduction in binding
affinity of ibrutinib to BTK.9 A potent noncovalent BTK
inhibitor, which does not utilize C481 covalent binding for
affinity, could still be efficacious for this portion of patients with
mutation. Several research groups have reported different
R
tissues, and other organs in the body. Initiation of the disease
involves the systematic dysregulation of T and B lymphocytes,
which leads to a breach of self-tolerance, resulting in immune
responses directed against self-antigens.1 Despite available
efficacious biological agents for the treatment of this condition,
not all patients tolerate or respond to those therapies, leaving
open the need for novel agents with different mechanisms of
action. Moreover, orally bioavailable small molecule drugs are
desirable in the treatment of RA as they offer an alternative to
parenteral administration of the biologic agent. The ability of
small molecule therapies to effectively treat rheumatoid arthritis
was demonstrated by Tofacitinib, a selective JAK inhibitor,
which achieved efficacy comparable to biological agents.2,3
Bruton’s tyrosine kinase (BTK) is a Tec family kinase
expressed in certain immune cells including B cells, mast cells,
and macrophages.4,5 It plays a critical role in multiple pathways
such as the B cell receptor (BCR) and Fcγ receptor (FcR)
signaling cascades, where it regulates the survival, activation,
proliferation, differentiation, and maturation of B cells. BTK’s
role in these pathways makes it a uniquely attractive target for
Received: December 1, 2015
Accepted: December 18, 2015
© XXXX American Chemical Society
A
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noncovalent binding BTK inhibitors.6 This letter describes the
discovery of the reversible noncovalent binding, potent and
selective BTK inhibitors based on 8-amino-imidazo[1,5-
a]pyrazines, exemplified by compounds 1−3 (Figure 1).
a
Scheme 2
a
Reagents. i. HATU, TEA, CH2Cl2, 0 °C; ii. POCl3, CH3CN, 57% two
steps; iii. NBS, DMF, rt, 95%; iv. 2 M NH3 in IPA, 120 °C, 97%.
to 13 in high yield with an ammonia solution, in isopropanol, at
120 °C in a sealed vessel overnight. SAR studies with different
substitutions on the piperidine ring were achieved with
substituted piperidine-3-carboxylic acids, which were either
commercially available or readily prepared.
Figure 1. Structures of ibrutinib and compounds 1−3.
Preparation of this series of BTK inhibitors is depicted in
Schemes 1 to 3. The modular nature of the synthetic route
Suzuki coupling (Scheme 3) of 7 and 13, using palladium
catalyst such as Pd(DPPF)CH2Cl2 and potassium carbonate 2
a
Scheme 1
a
Scheme 3
a
Reagents: i. (COCl)2, DMF,CH2Cl2, rt; ii. DIEA, DMAP, THF, 50
°C, 12 h, 77% two steps; iii. Bis(pinacolato) diboron, Pd(OAc)2,
XPhos, KOAc, dioxane, 90 °C, 6 h, 75%.
allows for quick SAR exploration around various parts of the
BTK inhibitor series. Scheme 1 depicts the preparation of the
boronic ester intermediate 7. 4-Bromo-3-fluorobenzoic acid 4
was converted to the intermediate benzoyl chloride by the
treatment with oxalyl chloride/DMF in dichloromethane, and
then further converted to 6 by coupling with 4-trifluoromethyl-
pyridine-2-amine 5 (diisopropylethylamine and a catalytic
amount of DMAP in acetonitrile). The aromatic bromine in
6 was then converted to boronic acid pinacol ester 7, by
reacting with bis(pinacolato) diboron, catalyzed by palladium
acetate with XPhos. Commercially available 4-bromobenzoic
acids, with different substitutions on the ring, facilitated SAR
development for different substitutions on the phenyl ring.
Similarly, when different substituted pyridine-2-amines or
heterocyclic amines are used for the synthesis in Scheme 1,
optimization of substitutions on the pyridine ring or the
heterocyclic replacements can be quickly accomplished.
The synthesis of intermediate 13 is depicted in Scheme 2.
Starting from (3-chloropyrazin-2-yl)methanamine 8, coupled
with (R)-N-Boc-piperidine-3-carboxylic acid 9, using HATU as
coupling reagent and triethylamine as the base, provided
compound 10. Cyclization of the amide in 10 to imidazole was
facilitated by treatment with phosphorus oxychloride in
acetonitrile at 80 °C for 5 h, followed by careful quenching
with 20% aqueous ammonium solution and crushed ice to
provide intermediate 11 in good yield. Bromination of
intermediate 11 was carried out using N-bromosuccinimide in
DMF at room temperature for 1 h, producing intermediate 12
in quantitative yield. The chloro intermediate 12 was converted
a
Reagents. i. Pd(dppf)CH2Cl2, 2 M K2CO3 (aq), dioxane, 60 °C, o/n,
80%; ii. TMSI, CH2Cl2, 5 °C, 99%; iii. HATU, DIEA, DMF, 0 °C, 1 h,
70%.
M aqueous solution in dioxane at 60 °C overnight provided a
good yield of product 14. In the following step, the Cbz group
was successfully removed with treatment of iodotrimethylsilane
in methylene chloride at low temperature in quantitative yield.
The free piperidine amine was then coupled with acids such as
3-methyloxytane-3-carboxylic acid 16, using HATU, with
Hunig’s base in DMF to provide the final product 2.10 SAR
for optimizing amide substitutions was done using different acid
to replace 16 at the final amide formation step.
All BTK inhibitors were evaluated in both a BTK enzymatic
assay11 and a human PBMC functional assay.11 SAR was carried
out on different areas of this series of BTK inhibitors based on
8-amino-imidazo[1,5-a]pyrazine core, using different starting
materials as indicated in the aforementioned synthesis schemes.
Table 1 describes the activities of compounds with different
aromatic amines, which make up the central amide. 2-Pyridyl
amides are more potent than other heteroaromatic amides such
B
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Table 1. SAR of the Amide Aromatic Amines
a
The data are average of at least two repeated tests. ND: not determined.
as pyrimidine (26, 27), pyrazine (28), pyridiazine (25), and
thioazole (29, 30). 4-Position substitutions on the 2-pyridyl
were best tolerated with different sizes and heteroatoms. These
structural changes also contributed to tuning the off target
activities and pharmacokinetic profiles of this series of
compounds while maintaining excellent potencies for the
BTK inhibition.
SAR with different substitutions on the middle phenyl ring
are described in Table 2. Compound 1 with no substitution has
excellent BTK inhibition potency. The 3-fluoro analogue (2) is
∼10× more potent than the 2-fluoro (31) and 2-chloro (32)
substitutions. However, compounds with a methoxy substitu-
tion on 2- and 3-positions (35 and 33) are equally potent. The
2,3-difluoro compound (36) has similar enzyme binding
activity as 2, but was approximately 2-fold less potent in the
cellular assay.
substitution provides the best BTK enzymatic and cellular
potency.
Variation of the piperidine amide was quickly explored by the
library synthesis approach of amide couplings. Functional
groups in different sizes were tolerated, showing excellent
potency in the enzyme assay. Thus, given the wide tolerance for
change, SAR in this area was applied to adjust the physical
chemical properties and off-target activities for this series. Table
4 lists 12 different amides on the piperidine (37, 3, 41−50),
with great BTK enzyme inhibition potency and cellular activity.
Based on the overall profile of this series, compounds 1, 2, and
3 were selected for further profiling because of their excellent
enzymatic and cellular potency.
The kinase selectivity profiles for compounds 1, 2, and 3
were accessed over the closely related Tec and Src family
kinases. Additionally, the activity profiles of compounds 1, 2,
and 3 were investigated by testing them at three concentrations
(1, 0.1, and 0.01 μM) against a commercially available panel of
265 human kinases.11 Ten point compound concentration
response curves were generated (at half-log intervals from 1
μM) for 17 kinases that demonstrated activity in the initial test.
Substitutions on the bottom piperidine ring were explored in
an attempt to increase the steric hindrance around the amide,
aiming to slow down the potential metabolic cleavage of the
amide (Table 3). Methyl (37), difluoromethyl (38), methox-
ymethyl (39), and trifluoromethyl (40) were applied. Methyl
C
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Table 2. SAR on Substitutions on the Phenyl Linker
Table 4. SAR of Amide Substitutions on Piperidine
a
a
compd
no.
BTK enzymatic assay IC50 hPBMC assay IC50
R1,R2
H, H
(nM)
(nM)
1
0.27
0.32
2.5
8.0
7.5
81
2
F, H
31
32
33
34
35
36
H, F
H, Cl
6.3
181
40
MeO, H
CF3O, H
H, MeO
F, F
1.1
38
ND
48
0.98
0.41
18
a
The data are average of at least two repeated tests. ND: not
determined.
Table 3. SAR of Substitutions on the Piperidine Ring
a
a
BTK enzymatic assay IC50 hPBMC assay IC50
compd
R1,R2
H, H
(nM)
(nM)
1
0.27
0.17
0.86
0.31
3.7
8.0
3.0
20
a
The data are average of at least two repeated tests.
37
38
39
40
F, Me
H, CF2H
H, MeOCH2
F, CF3
Table 5. Selectivity of 1, 2, and 3 over SRC Family Kinases
4.0
ND
compd 1 IC50 (nM) compd 2 IC50 (nM) compd 3 IC50 (nM)
a
kinase
(fold selectivity)
(fold selectivity)
(fold selectivity)
The data are average of at least two repeated tests. ND: not
determined.
BTK
BLK
0.27
0.32
0.31
84 (311)
28 (104)
ND
42 (131)
20 (62)
20 (65)
BMX
CSK
11 (34)
A summary of data for this kinase screen set is shown in Table
5. Fold selectivity over BTK enzyme is in parentheses. The
selectivity ratios against these kinases, most from the Tec and
Src family, are greater than 50-fold, except for compound 3,
which was 34-fold for BMX and 38-fold for LCK.
253 (791)
81 (253)
55 (172)
187 (584)
322 (1006)
921 (2878)
22 (69)
201 (648)
65 (203)
37 (116)
111 (347)
198 (619)
1200 (3750)
12 (38)
ERBB4
FGR
FRK
118 (437)
ND
361 (1337)
ND
FYN
ITK
Cocrystals of compounds 1 and 3 with BTK were obtained.12
The key interactions of compound 1 with BTK are shown in
Figure 2A. The 8-amino and 7-nitrogen on the imidazo[1,5-
a]pyrazine each make one hydrogen bond with the BTK hinge
region, and the primary amine is also within H-bonding
distance to the gatekeeper T474 side-chain alcohol, while the 2-
nitrogen forms a hydrogen bond with a water molecule. The
amide carbonyl, on the piperidine ring, is oriented for a H-bond
with the amide nitrogen of peptide G480 and C-481. A key
interaction of the 2-amino-pyridine is a bidentate hydrogen
bond interaction with S538 side chain hydroxy and the amide
nitrogen of D539. The trifluoromethyl and the pyridine ring are
in a hydrophobic pocket with great flexibility, which could be
ND
LCK
LYNB
PTK6
SRC
36 (133)
399 (1478)
66 (244)
ND
184 (575)
63 (197)
116 (362)
24 (75)
131 (423)
60 (194)
89 (287)
23 (74)
SRMS
TEC
TXK
YES1
32 (119)
167 (619)
49 (181)
ND
69 (216)
32 (100)
43 (134)
63 (203)
33 (106)
21 (68)
unique to the BTK enzyme. The inhibitor takes advantage of
the wide access to the back pocket because of the smaller
D
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Figure 2. (A) X-ray crystal structure of 1 in the active site of BTK enzyme with hydrogen bond interactions to the key residues. (B) The overlay of
X-ray crystal structures of 1 (green) and 3 (purple) in the active site of BTK enzyme showing the flexibility of the enzyme for the hydrophobic
pocket around the trifluoromethylpyridine with significant movement of F442 and F413.
threonine gatekeeper. Overlay of the X-ray crystal structures of
1 and 3 reveals significant differences in binding mode around
the trifluoromethylpyridine. There are no hydrogen bond
interactions of 3 at the aminopyridine with the S538 and D539.
The trifluoromethylpyridine ring extends deeper into the back
pocket with the side chain of F442 and F413 moving away to
accommodate a different binding mode in the complex with 3.
This binding into the deep back pocket compensates the loss of
interaction of the aminopyridine bidentate hydrogen bond. In
the mean time, significant away movement of the loop close to
the axial methyl substitution on the piperidine ring for
compound 3 is noticed, which allows compound 3 to slide
deeper into the pocket. The flexibility of the BTK enzyme in
this region allows quite broad SAR with substitutions of various
sizes (at the pyridine 4-position) to fit in the back pocket as we
described before, allowing the optimization of potency and
kinase selectivity. All these interactions provide this series of
BTK inhibitors with their potency and selectivity.
Table 6. Pharmacokinetic Parameters of Compounds 1, 2,
and 3
compd
1
2
3
IV
dose
PO
dose
IV
dose
PO
dose
IV
dose
PO
dose
rat
dose (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
AUCN (μM·h)
T1/2 (h)
4
10
2
5
2
5
6.3
0.59
5.1
1.2
24
1.7
1.1
1.4
18
2.6
1.6
1.8
Cmax (μM)
Tmax (h)
4.8
2.2
84
1.0
0.5
77
0.40
1.0
22
F (%)
IV
dose
PO
dose
IV
dose
PO
dose
IV
dose
PO
dose
dog
dose (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
AUCN (μM·h)
T1/2 (h)
1
2
1
2
11
2.0
3.0
4.1
1.5
0.65
20
Compounds 1, 2, and 3 were evaluated in a human whole
blood assay that assessed the effect of compounds on the
upregulation of CD69 on B cells in response to BCR-cross-
linking by an anti-CD79b antibody.11 Compounds 1, 2, and 3
all demonstrated excellent activity in the human whole blood
assay with IC50 of 120 (n = 2), 113 (n = 11), and 94 (n = 11)
nM, respectively.
6.5
Cmax (μM)
Tmax (h)
0.78
1.0
60
3.9
2.0
82
F (%)
In rat pharmacokinetic studies, compounds 1, 2, and 3
exhibited reasonable exposure, clearance, half-life, and bio-
availabilities (Table 6). Additionally, compounds 2 and 3 were
evaluated in dog pharmacokinetic studies, and compound 3
showed higher drug levels with a longer half-life compared to
compound 2.
Compound 3 was tested for efficacy in the rat model of
collagen-induced arthritis (CIA).11 In a prophylactic treatment
protocol, compound 3 demonstrated a dose-dependent (1, 3,
10, and 30 mg/kg, PO, QD) decrease in paw thickness, as
indicated by significant reduction (p < 0.0001) of paw thickness
through day 30 consequent to CIA induction (Figure 3).
In summary, we described the identification of reversible
BTK inhibitors based on the 8-aminoimidazo[1,5-a]pyrazine
Figure 3. Dose-dependent changes in paw thickness in a rat CIA
model at different doses of 3.
E
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Kinase Inhibitors: Approaches to Potent and Selective Inhibition,
Preclinical and Clinical Evalution for Inflammatory Diseases and B
Cell Malignancies. J. Med. Chem. 2012, 55, 4539−4450.
core. The inhibitors are characterized by high enzymatic
potency, selectivity over other kinases, and acceptable oral
bioavailability. SAR studies of the different parts of this series of
inhibitors led to the identification of several lead compounds
such as 1, 2, and 3. Further characterization by X-ray crystal
structures revealed specific interactions between the inhibitors
and the BTK enzyme, which likely account for the observed
BTK inhibition activity and selectivity over other kinases.
Compound 3 combines an optimization of the BTK enzyme
binding, cellular functional activity, human whole blood assay
activity, kinase selectivity, and in vivo pharmacokinetic profile.
This orally available noncovalent BTK inhibitor 3 displays
efficacy in reducing paw thickness in a rat model of collagen-
induced arthritis. Further investigation of the SAR of this series
of BTK inhibitors will be the subject of another report.
(7) Cameron, F.; Sanford, M. Ibrutinib: First Global Approval. Drugs
2014, 74 (2), 263−271.
(8) Chang, B. Y.; Huang, M. M.; Francesco, M.; Chen, J.; Sokolove,
J.; Magadala, P.; Robinson, W. H.; Buggy, J. J. The Bruton tyrosine
kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by
inhibition of multiple effector cells. Arthritis Research & Therapy
2011, 13 (4), R115.
(9) 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.; Byrd, J. C.
Resistance Mechanisms for Bruton’s Tyrosine Kinase Inhibitor
Ibrutinib. N. Engl. J. Med. 2014, 370, 2286−2294.
(10) The detailed synthetic procedures and characterization of the
key compounds can be found in the Supporting Information.
(11) The protocol for the following assays are described in the
Supporting Information: BTK enzymatic binding assay; BTK human
PMBC functional assay; kinase panel selectivity assay; BTK human
whole blood assay; the rat model of collagen-induced arthritis.
(12) X-ray crystal methods will be published elsewhere. The
coordinations of the X-ray crystal structures for compounds 1 and 3
have been published on protein data bank with codes 5FBN and
5FBO, respectively.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00463.
■
S
Synthetic methods, characterization of key compounds,
and biology assay protocols (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: 1-732-594-9600. E-mail: jian_liu@merck.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Wilfredo Pinto at Merck Research Laboratories
for measuring the high-resolution mass and Dr. Lu Yang for
1
taking HNMR and 13CNMR for key compounds.
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