Discovery of 4-Aminoquinoline-3-carboxamide derivatives as potent reversible Bruton's tyrosine kinase inhibitors for the treatment of rheumatoid arthritis

Article abstract of DOI:10.1021/acs.jmedchem.9b00329

A structure-hopping strategy was applied to discover a series of novel 4-aminoquinoline-3-carboxamide derivatives as potent, reversible BTK inhibitors. Compared to the previously described cinnoline scaffold compounds, the 4-aminoquinoline analogues showed significantly improved drug-like properties, especially in their aqueous solubility. The most potent compound, 25, displayed a stronger inhibitory effect on both BTK^WT (IC₅₀ = 5.3 nM) and BTK^C481S (IC₅₀ = 39 nM). In a rodent collagen-induced arthritis model, compound 25 efficiently reduced paw swelling without a loss in body weight. On the basis of potency, drug-like properties, stability, and noncovalent mode of inhibition, our representative inhibitors could have a promising profile to be treatments for a wide range of autoimmune diseases.

Full text of DOI:10.1021/acs.jmedchem.9b00329

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Article
Discovery of 4-Aminoquinoline-3-carboxamide Derivatives
as Potent Reversible Bruton's Tyrosine Kinase
Inhibitors for the Treatment of Rheumatoid Arthritis
Xia Yao, Xiuyun Sun, Shuyu Jin, Ling Yang, Hongjiang Xu, and Yu Rao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00329 • Publication Date (Web): 01 Jul 2019
Downloaded from http://pubs.acs.org on July 1, 2019
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Discovery of 4-Aminoquinoline-3-carboxamide
Derivatives as Potent Reversible Bruton's Tyrosine
Kinase Inhibitors for the Treatment of Rheumatoid
Arthritis
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Xia Yao,1 Xiuyun Sun,1,2, ‡ Shuyu Jin, Ling Yang, Hongjiang Xu, and Yu Rao
, ‡
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4
4
1, 
1Ministry of Education (MOE) Key Laboratory of Protein Sciences, School of
Pharmaceutical Sciences, MOE Key Laboratory of Bioorganic Phosphorus Chemistry &
Chemical Biology, Tsinghua University, Beijing 100084, P.R. China.
2Tsinghua-Peking Center for Life Sciences, Beijing 100084, P.R. China.
3Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, P.R. China.

Corresponding author. e-mail: yrao@tsinghua.edu.cn
These authors contributed equally to this manuscript.
‡
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4R&D Institute, Chia Tai Tianqing Pharmaceutical Group Co.,LTD, Nanjing 210023, P.R.
China.
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KEYWORDS. Structure hopping, Reversible BTK inhibitors, 4-Aminoquinoline-3-
carboxamide derivatives, Rheumatoid arthritis
F
NH2
N
O
NH2
N
O
NH2
NH2
O
NH2
NH2
Structure
Hopping
Structure-based
optimization
H CO
3
N
N
H C
3
H3C
NH
N
NH
N
NH
N
compd.25
BTK IC50 = 5.3 nM
BTK C481S IC50 = 39 nM
Aq. Sol. = 7.3 g/mL
compd.6
cinnoline scaffold
Aq. Sol. < 0.05 g/mL
compd.7
quinoline scaffold
Aq. Sol. = 10.5 g/mL
Solubility improved > 200 fold
In vivo anti-RA
Plasma recovery % (human,rat,
monkey,mouse,dog) > 88%
ABSTRACT. A structure-hopping strategy was applied to discover a series of novel 4-
aminoquinoline-3-carboxamide derivatives as potent, reversible BTK inhibitors.
Compared to the previously described cinnoline scaffold compounds, the 4-
aminoquinoline analogs showed significantly improved drug-like properties, especially in
their aqueous solubility. The most potent compound, 25, displayed a stronger inhibitory
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effect on both BTKWT (IC = 5.3 nM) and BTK
C481S
(IC = 39 nM). In a rodent collagen-
50
50
induced arthritis model, compound 25 efficiently reduced paw swelling without a loss in
body weight. On the basis of potency, drug-like properties, stability and noncovalent
mode of inhibition, our representative inhibitors could have a promising profile to be
treatments for a wide range of autoimmune diseases.
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INTRODUCTION.
Rheumatoid arthritis (RA) is a multifactorial autoimmune disease that can cause serious
1
cartilage destruction and bone erosion. Therapeutic medications for RA include
nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs
(
DMARDs), TNF alpha inhibitors, IL-6 inhibitors, T-cell activation inhibitors, B-cell
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depleters, immunosuppressants, etc. Tofacitinib, a JAK1/3 inhibitor, was approved by
the FDA in 2012 and served as the first kinase inhibitor for the treatment of autoimmune
diseases. It has been reported that tofacitinib has similar efficacy to adalimumab in RA
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patients who show a poor response to methotrexate (MTX) administration. The
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success of tofacitinib motivated us to search for other kinase targets that are implicated
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in RA. Recently, several findings suggested that inhibiting the activity of Bruton’s
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tyrosine kinase (BTK) could provide a promising treatment profile for autoimmune
diseases such as RA and systemic lupus erythematosus (SLE).5,6 BTK belongs to the
Tec non-receptor tyrosine kinase family and participates in various signal pathways,
including B cell receptor (BCR) pathway. It is well known that BTK plays an important
role in the differentiation, activation, proliferation and survival of B lymphocytes.7-9
Studies in rodent arthritis models have provided evidence for a dual mechanism of
action: (i) inhibition of BCR-dependent B lymphocyte activation and autoantibody
secretion and (ii) suppression of myeloid cell-dependent inflammatory cytokine
production.10,11
For over a decade, several BTK inhibitors have been discovered for clinical therapy in
hematologic malignancies and chronic inflammatory diseases.12-15 Among these
molecules, Ibrutinib (Figure 1, 1),16,17 the most advanced BTK inhibitor, has been
approved for B cell malignancies and chronic lymphocytic leukemia.18-22 Ibrutinib is an
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irreversible inhibitor of BTK that covalently binds to Cys481 in the kinase domain.
Though covalent BTK inhibitors, such as spebrutinib (CC-292. Figure 1, 2)23 and
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acalabrutinib (ACP-196. Figure 1, 3),24,25 are currently involved in clinical development
for RA, these molecules actually possess many safety risks. For instance, the Michael
receptor portions of covalent inhibitors raise the concern of unwanted off-target
inhibition.26 In addition, haptenization of serum proteins by reactive groups potentially
leads to allergic reactions in patients, such as fever, lymphadenopathy, edema and
albuminuria. It is widely acknowledged that reversible inhibitors are more likely to
provide a lower risk of toxicity in RA patients compared to an irreversible inhibitor.
However, compared to covalent inhibitors, the development of reversible BTK inhibitors
has been slow.27-46 To date, no reversible inhibitors have been approved for clinical
applications. The well-known noncovalent BTK inhibitors, RN-486 (Figure 1, 4) and
GDC-0834 (Figure 1, 5), both suffer from poor stability and pharmacokinetic (PK)
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profiles. Considering the prospects of BTK for the treatment of autoimmune disorders,
the development of novel reversible inhibitors with favorable drug-like properties is
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urgently needed. In this work, a structure-hopping strategy was applied to discover 4-
aminoquinoline-3-carboxamide derivatives as potent BTK inhibitors. Removal of the
insignificant nitrogen and incorporation of methoxy and fluorine group substitutions
significantly improved the aqueous solubility without compromising the activity against
BTK. The most potent inhibitor exhibited strong inhibition of BTK and cellular Tyr223
autophosphorylation. In addition, the modeling of compound 25 in the active site of BTK
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suggested that the binding pose of our reversible inhibitor was orthogonal to that of
C481S
Ibrutinib. As a result, 25 exhibited an IC value of 39 nM against the BTK
protein
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and provided improved kinase selectivity when compared to Ibrutinib, particularly
against EGFR, TEC, and ITK.17,47-49 In a rodent collagen-induced arthritis (CIA) model,
compound 25 efficiently reduced paw swelling without a loss in body weight, implying
that our inhibitor is efficacious and well tolerated in this study.
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O
H
N
N
O
NH2
N
F
N
NH2
N
N
N
N
N
NH
H
O
N
O
N
N
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, Ibrutinib
2, Spebrutinib (CC-292)
3, Acalabrutinib (ACP-196)
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OH
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, RN-486
5, GDC-0834
Figure 1. Well-known BTK inhibitors.
RESULTS AND DISCUSSION.
Design and in vitro study. Previously, compound 6 was reported as a potent reversible
BTK inhibitor.35 While it demonstrated good in vitro activity against BTK, compound 6
exhibited poor aqueous solubility (at pH 7.4 < 0.05 μg/mL), which limited its in vivo
study. Given the stability and inhibitory activity of compound 6, we envisioned that
optimization might yield a promising lead candidate with improved physiochemical
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properties with a retention in potency. It is well acknowledged that compounds with
suitable aqueous solubility and CLogP values are more likely to provide acceptable PK
properties for oral administration.50
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Our preliminary studies suggested that the cinnoline moiety might be the source of the
poor solubility observed for compound 6. By analyzing the binding mode of 6 (PDB code:
4Z3V), we found that the cinnoline-N2 atoms did not make direct H-bond interactions
with the kinase domain. Based on this observation, the initial design was to remove the
insignificant nitrogen and introduce various substitutions, which aimed to disrupt the
molecular planarity and lead to additional interactions with the protein.
To our delight, compared to compound 6, compound 7 bearing a 4-aminoquinoline-3-
carboxamide core improved the aqueous solubility by more than 200-fold but with a
significant decrease in inhibitory activity. Additionally, compound 7 exhibited a suitable
CLogP value, consistent with our drug-like physicochemical criteria. However,
replacement of the cinnoline moiety with 4-hydroxyquinoline-3-carboxamide (8) resulted
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in a dramatic loss in BTK inhibition, indicating that the 4-NH group is important for
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ligand binding (Table 1).
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The docking pose of compound 7 bound to the ATP site of BTK suggested that the 4-
aminoquinoline-3-carboxamide moiety would form three direct and one water-mediated
H-bond interaction with the hinge region of BTK. The indazole moiety stabilized the
conformation of the P-loop by forming crucial hydrogen bonds with Phe413 and Gly414
(Figure 2A). Compared to RN-486, compound 7 lacked the hydrophobic interaction with
the H3 pocket of BTK, which induced a significant conformational change in BTK such
that the key activating tyrosine, Tyr551, was unphosphorylated and sequestered from
solvent (Figure 2B). It has been reported that the H3 pocket is responsible for the
exquisite selectivity of the well-known reversible BTK inhibitor CGI-1746.28 Based on
these observations, the first design was to compensate for the hydrophobic interaction
with the H3 pocket of the quinoline compounds.
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Table 1. In vitro BTK inhibition, aqueous solubility and CLogP value
BTK Inhibition
(% at 100 nM)a
Aq. solubility
(μg/mL)b
CLogPc
Compd.
6
Structure
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NH2
O
NH2
NH2
NH2
N
N
92.1 ± 3.9
54.2 ± 0.4
15.5 ± 2.8
<0.05
10.5
n.d.
2.51
H C
3
NH
N
NH2
N
O
7
8
3.07
4.04
H C
3
NH
N
OH
N
O
H C
3
NH
N
a. BTK inhibition (%) by the compound at 100 nM, data represents as mean ± SD of
triplicates. b. Kinetic solubility (μg/mL) in PBS (pH 7.4) at 25 °C, determined by HPLC,
n=3. c. Values are predicted by ChemBioDraw software Ultra 14.0.
Because the methyl substituent of compound 7 was proposed to have hydrophobic
interactions with Leu528, the quinoline and indazole rings were locked at a dihedral
angle of 104°. To increase the possibility that the extended groups could approach the
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H3 pocket, the methyl group should be removed. As shown in Table 2, compound 9
without the methyl substituent inhibited BTK 41.6% at 100 nM, which was less potent
than compound 7. The hydrophobic phenoxyl, phenylamino, benzyloxyl, and
benzyl(methyl)amino groups provided obvious changes in the overall lipophilicity and
significantly decreased the inhibitory activity (10-13). Similarly, the 1,1'-biphenyl-4-yloxyl,
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1,1'-biphenyl-3-yloxyl and 4-(4-phenoxyphenoxyl) substituted compounds showed very
weak inhibition of BTK kinase (14-16). We suspected that our designed compounds
failed to induce the formation of the H3 pocket; instead, the substituted groups
increased the steric bulk and prohibited the inhibitors from accessing to the ATP binding
site of BTK.
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9
0
Figure 2. (A) Putative binding mode of compound 7. The red arrow points out the
extended orientation for the hydrophobic groups to grow toward the H3 pocket. Selected
residues are displayed in stick representation. Orange dashes represent hydrogen
bonds. (B) Docking structure of BTK bound to compound 7 (green and blue)
superimposed on X-ray cocrystal structure of BTK bound to RN-486 (PDB code: 4OTR,
gray and yellow). RN-486 had hydrophobic interaction with H3 pocket which induced a
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significant position change of key activating tyrosine Tyr551 such that it was in the
unphosphorylated state and sequestered from solvent.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Enzymatic BTK potency of compounds bearing lipophilic groups on the
indazole ring
NH2
N
O
NH2
R1
NH
N
BTK Inhibition
CLogPb
2.87
Compd.
9
R1
% at 100 nM)a
(
H
41.6 ± 0.3
O
1
0
1
3.9 ± 1.3
10.4 ± 6.2
9.5 ± 0.7
24.7 ± 0.9
10.7 ± 2.8
4.96
H
N
1
4.88
O
12
4.97
N
1
3
4
5.19
O
1
6.85
Ph
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Ph
O
1
5
n.a.
6.85
7.06
O
16
11.8 ± 3.5
PhO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a. BTK inhibition (%) by the compound at 100 nM, data represents as mean ± SD of
triplicates. n.a. means no activity. b. Values are predicted by ChemBioDraw software
Ultra 14.0
Our second optimization focused on introducing additional interactions with the protein,
seeking highly potent inhibitors with a minimal change to the lipophilicity. Quite often,
fluorine is used to modulate physicochemical properties, improve metabolic stability,
5
1
and enhance binding affinity to the target protein. As shown in Table 3, compound 17,
with a 5-F substitution, exhibited a significantly improved effect on BTK, which
maintained a substantial amount of the BTK inhibition but potentially increased the
aqueous solubility profile relative to 6. Furthermore, we explored the potency of the
compounds with 6-F, 6-NO and 6-NH substitutions (18-20), but only compound 20
2
2
displayed an acceptable inhibition rate of 36.5% inhibition at a concentration of 20 nM.
In addition, replacement of the indazole by a benzimidazolone ring resulted in a loss of
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affinity for BTK (21-24), which might be attributed to the missing π-stacking interaction
with the P-loop. Finally, we kept the 5-F substitution and replaced the methyl group with
a methoxy group on the indazole ring. The resulting compound 25 exhibited 68.7%
inhibition at a concentration of 20 nM, which might possess higher potency and more
favorable drug-like properties than the lead compound.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inspired by the improved activity of the 4-aminoquinoline-3-carboxamide derivatives, the
selected compounds were further assessed by their IC values against BTK and kinetic
50
aqueous solubility profiles. As shown in Table 4, the compounds bearing a quinoline
core showed over a 100-fold improvement in aqueous solubility, supporting our initial
strategy. The 5-F substitution on the quinoline ring exhibited a 6-fold increase in
potency (17, IC50 of 11.6 nM; 7, IC50 of 72.8 nM). Compound 25 was found to be
equipotent to compound 6 but with significantly improved aqueous solubility (at pH 7.4 =
7.3 μg/mL), which was more advantageous for further in vivo tests.
Table 3. In vitro potency of 4-aminoquinoline-3-carboxamide derivatives
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NH2
O
R2
5
6
NH2
3
7
N
1
R3
CLogPb
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compd.
R2
R3
BTK Inhibition
(
% at 20 nM)a
45.9 ± 1.2
H
N
1
7
8
5-F
3.25
N
N
N
N
O
O
H C
3
H
N
1
6-F
6-NO2
6-NH2
5-F
25.3 ± 11.8
12.8 ± 7.7
36.5 ± 6.7
9.3 ± 2.8
5.1 ± 3.8
n.a.
3.25
2.92
2.52
2.55
2.55
2.75
2.75
H C
3
H
N
19
20
21
22
23
24
H C
3
H
N
H C
3
H
N
N
H
H
N
6-F
N
H
H
N
5-F
O
O
N
H
H
N
H C
3
6-F
10.0 ± 5.9
N
H
H C
3
H
N
25
5-F
-
68.7 ± 1.3
44.6 ± 4.6
2.83
2.51
N
H CO
3
6
-
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a. BTK inhibition (%) by the compound at 20 nM, data represents as mean ± SD of
triplicates. n.a. means no activity. b. Values are predicted by ChemBioDraw software
Ultra 14.0.
Table 4. IC values and aqueous solubility profiles
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Aq. solubility (μg/mL)b
Compd.
7
BTK IC (nM)
50
72.8 ± 0.9
11.6 ± 0.7
5.3 ± 1.3
5.6 ± 1.0
10.5
5.2
17
25
6
7.3
<0.05
a. Data is mean ± SD of three independent experiments. b. Kinetic
solubility (μg/mL) in PBS (pH 7.4) at 25 °C, determined by HPLC,
n=3.
In the BCR signaling pathway, BTK Tyr551 transphosphorylation is followed by the
autophosphorylation of Tyr223 in the SH3 domain. In this way, BTK is activated and
transphosphorylates the downstream substrate PLCγ2. Given their good kinase activity,
selected compounds were further evaluated for their inhibition of cellular
autophosphorylation in Ramos cells. Herein, the phosphorylation levels of BTK (Tyr223)
and total BTK protein levels were analyzed by western blotting with anti-pY223 or anti-
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1
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
total BTK antibodies. As shown in Figure 3A, compounds 6, 17 and 25 all exhibited
dose-dependent inhibition and efficiently blocked Tyr223 autophosphorylation at a
concentration of 1 μM. As expected, inhibitor 25 (EC50 of 42.7 nM) was 2.4-fold more
potent than 6 (EC50 of 100.9 nM) in cell-based assays (Figure 3B), which may be
derived from the better aqueous solubility and cell permeability of the quinoline core.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Figure 3. BTK inhibitors blocked autophosphorylation of Tyr223. (A) Concentration-
dependent inhibition by compound 6, 17, 25 in Ramos cells of pervanadate mediated
phosphorylation of BTK Tyr223. The phosphorylation levels of BTK (Tyr223) and total
BTK protein levels were analyzed by western blotting. (B) EC curves of compound 25
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
and 6. Data is mean ± SD of three independent experiments.
Docking study and selectivity profile. To better understand why the substitution of
fluorine and methoxy groups resulted in more than a 10-fold improvement in potency
compared to compound 7 (7, IC50 = 72.8 nM; 25, IC50 = 5.3 nM), we performed
molecular docking studies on compounds 7 and 25. As shown in Figure 4A, when
properly aligned, compound 25 appeared to situate into a space that was slightly
different from that of 7. The incorporation of the fluorine and methoxy substitutions likely
tuned the conformation, altering the dihedral angle between the quinoline and indazole
rings from 104° in compound 7 to 100° in compound 25. In addition, the fluorine atom
was proposed to add a hydrophobic interaction with Leu528. The movement of the
indazole ring allowed 25 to form stable π–cation interactions with Lys430 (Figure 4B).
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1
1
2
2
2
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The modeling of our inhibitors in the active site of BTK also suggested that the binding
pose of compound 25 was orthogonal to that of Ibrutinib (Figure 5A). Compound 25 did
not interact with Cys481 that was responsible for the covalent interaction of Ibrutinib.
Given that the BTKC481S mutation is prevalent in patients who relapse after Ibrutinib
treatment, we hypothesized that our inhibitor could retain activity against the C481S
mutant form of BTK. Herein, ACP-196 was selected as a control compound, which is a
second-generation covalent inhibitor of BTK, with higher potency and less off-target
effects than Ibrutinib. As expected, 25 efficiently inhibited BTKC481S with an IC value of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
3
9 nM. In contrast, ACP-196 only showed equivalent potency to 25 against BTKWT but
failed to block the phosphorylation effect of BTKC481S. In addition, we tested the effects
of 25 and ACP-196 against EGFR and TEC, two well-known off-targets of Ibrutinib. To
our delight, 25 and ACP-196 had no effect on EGFR, and their IC values were above
50
2
μM. For TEC kinase, 25 was less potent than ACP-196. The selectivity of 25 for
BTKWT over TEC was more than 40-fold (Figure 5B). Inspired by the preliminary
selectivity data, a kinase selectivity profile for compound 25 was further assessed for
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the closely related kinases from the TEC and SRC families (Table 5). Based on the
determined IC values, compound 25 was more selective for BTK than other members
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of the TEC and SRC kinase families. The selectivity ratios were all greater than 30-fold,
except for BLK (28-fold). It is of note that compound 25 was 58-fold less potent against
ITK, which is highly homologous to BTK but positively regulates the T cell receptor
signaling pathway and acts as a crucial regulator in multiple physiological processes of
52
T lymphocytes. The selectivity data indicated that 25 might have a slight effect on the
physiological process of T cells. Above all, it was envisioned that our reversible inhibitor
would have fewer side effects than the covalent BTK inhibitors. Because 25 could have
a profile to provide potential treatment for patients with B cell malignancies harboring
the BTKC481S mutation with less off-target effects, this compound is currently being
evaluated in Ibrutinib-resistant NHL and CLL studies.
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Docking study of 4-aminoquinoline-3-carboxamide derivatives. (A) Putative
binding modes of compound 7 (orange) and 25 (green) in the BTK kinase domain
(yellow cartoon). Selected residues are shown in stick representation. (B) Summary of
receptor interactions with compound 25. Dark blue ball (positive charged interaction);
cyan and red ball (polar interaction); green ball (hydrophobic interaction); purple arrow
(H-bond); red line (π–cation interaction).
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Compound 25 binds to the kinase domain of BTK with a different manner of
the existing ATP-mimetic covalent inhibitors. (A) Superposition of the cocrystal structure
of Ibrutinib (orange, PDB code: 5P9J) and docking pose of 25 (blue). Ibrutinib forms a
covalent bond with residue Cys481 and occupies the hydrophobic pocket behind the
gatekeeper residue Thr474, 25 does neither. Residue Cys481 is shown in stick
representation. (B) IC50 values of 25 and ACP-196 against BTK, BTKC481S, EGFR and
TEC kinases.
Table 5. Kinase selectivity profile of compound 25
a
Selectivity ratiob
Kinase Kinase family
IC (nM)
50
BTK
ITK
TEC
TEC
5.3
306.9
/
58
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TEC
BLK
SRC
FYN
YES
TEC
SRC
SRC
SRC
SRC
227.0
43
28
150.2
902.5
170
31
165.2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>10000
>1000
a. Run in triplicates. b. Kinase of interest IC /BTK IC .
5
0
50
Plasma stability and in vivo PK profile. Inspired by the high potency and selectivity
profile, we further evaluated the drug-like properties of compound 25 with a plasma
incubation assay and a rat pharmacokinetic study. Determination of the plasma
recovery rate revealed the remarkable stability of our BTK inhibitor. The percent of the
remaining parent compound was above 88% after incubation in plasma samples from
humans, dogs, rats, mice and monkeys at 37°C for up to 2 h. In addition, no significant
interspecies differences were observed (Table 6). Compound 25 also exhibited a
favorable PK profile in rats. After a single 2 mg/kg iv dose, the mean clearance rate (CL)
and half-life (t ) of compound 25 were 20.8 mL/min/kg and 1.2 h, respectively.
1/2
Meanwhile, acceptable absorption properties were demonstrated after oral
administration at a dose of 5 mg/kg. Compound 25 showed a moderate oral
bioavailability (F) of 25% (Table 7).
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Table 6. In vitro plasma stability of compound 25a
Compd.
Human (%)
Dog (%)
Rat (%)
Mouse (%) Monkey (%)
98% 88%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
25
93%
88%
96%
o
a. % of remaining parent compound after 2 h incubation in plasma at 37 C with initial
concentration of 1 μM.
Table 7. Pharmacokinetic profile of compound 25 in rata
Compd.
CL (mL/min/kg), iv
V (L/kg)
d
T (h), iv
1/2
F (%)
2
5
20.8
2.1
1.2
25
a. Male Sprague-Dawley rats iv at 2 mg/kg (10% DMSO, 90% PEG400) and po at 5
mg/kg (0.5% methylcellulose suspension), n=6.
In vivo CIA study. Given the high in vitro potency and favorable PK profile of compound
25, we further evaluated the potential of our reversible inhibitor to treat RA using a
rodent collagen-induced arthritis (CIA) model. Arthritis was established in male DBA/1
mice by injecting type II collagen twice separately on day 0 and day 20 of the study. On
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day 29, the CIA model mice were randomized and treated orally with 0.2 mg/kg
dexamethasone (DXMS), 10 mg/kg compound 25 or vehicle control QD. The paw
volumes were then measured on days 29, 33, 36, 40, and 43. From day 29 to day 36, in
vehicle-treated mice, there was a dramatic increase in the paw volume, indicating that
the arthritis model was successfully established. Compared to the vehicle group, the
mice that were administered 25 or DXMS did not show disease progression.
Furthermore, the paw volumes of the drug-treated groups significantly decreased over
the duration of treatment. The QD 10 mg/kg dosage of 25 showed comparable efficacy
relative to DXMS (Figure 6A). During the study period, the body weights of the mice in
these four groups were recorded to evaluate the toxicity of the test compounds. As
shown in Figure 6B, the mice in the 25-treated group had a better appetite than those
treated with DXMS, which implied that our inhibitor may have lower side effects in vivo.
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Figure 6. Efficacy of compound 25 in rodent CIA model. (A) The paw swelling rate of
four groups, including vehicle, 25 (10 mg/kg, QD), DXMS (0.2 mg/kg, QD) and naive,
male DBA/1 mice, n=6. Data represent as mean ± SEM in the graphs. P < 0.001, ****P
***
<
0.0001 vs vehicle control by Dunnett’s test. (B) The average body weights of the mice
over study days.
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6
Chemistry. All of the title molecules 7-25 were prepared with a convergent route as
depicted in Schemes 1-4. Scheme 1 shows the preparation of the quinoline moieties,
intermediates 30a-d. The corresponding anilines and diethyl ethoxymethylenemalonate
were heated at 100°C for 2 h. After completion of the reaction, the mixtures were added
into boiling DOWTHERM A. Cyclization was accomplished under high temperatures to
give quinolones 27a-d. After hydrolysis, one-pot reactions by the addition of SOCl2
followed by ammonium solution furnished compounds 29a-d. Then, intermediates 30a-d
0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
were finally accomplished by treating 29a-d with 7 M NH in MeOH. Notably, the
3
products that resulted from these four steps were purified by filtration without column
chromatography, simplifying and expediting the synthesis.
Scheme 1. Synthesis of quinoline intermediatesa
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O
O
O
O
R2
R2
R2
OEt
OH
(
a)
(b)
NH2
N
H
N
H
Br
Br
Br
2
6a-d
27a-d
28a-d
1
1
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1
1
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0
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0
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9
0
Cl
O
NH2 O
R2
R2
NH2
NH2
N
N
(c)
(d)
Br
9a-d
Br
30a-d
2
a

Reagents and conditions: (a) (i) diethyl ethoxymethylenemalonate, 100 C, 2 h, (ii) Dowtherm
A, 250 C 4-12 h, 40-60% (b) LiOH, THF/EtOH/H O= 4:4:2, 55 C, 4-8 h, 75-82%; (c) (i)

2


SOCl , DMF, 70 C, 3-6 h, (ii) NH ·H O, DCM, 0 C - rt, 1 h, 40-60%; (d) 7M NH in MeOH, 80
2
3
2
3
C, overnight, 50-55%.
The synthesis of title compounds 9-16 is shown in Scheme 2. Starting from 1-bromo-
3
,5-difluorobenzene 31, after treatment with LDA and DMF, the aldehyde intermediate
32 was obtained. Then the intermediates 33b-h were furnished by an SNAR reaction and
converted into indazoles via the addition of hydrazinehydrate. Specifically, the synthesis
of compound 9 was started from the commercially available 4-bromo-2-
fluorobenzaldehyde 33a. Under strongly basic conditions, the brominated indazoles
were transformed into their corresponding borate intermediates 35a-h. The final step
was a Suzuki couplings, which worked well under microwave conditions.
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