Discovery of 4,7-Diamino-5-(4-phenoxyphenyl)-6-methylene-pyrimido[5,4- b]pyrrolizines as Novel Bruton's Tyrosine Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00441

An alternative medicinal chemistry approach was conducted on Bruton's tyrosine kinase (BTK) inhibitor 1 (ibrutinib) by merging the pyrazolo[3,4-d]pyrimidine component into a tricyclic skeleton. Two types of compounds were prepared, and their biochemical activities on BTK as well as stereochemistry effects were determined. Structural optimization focusing on the reactive binding group to BTK Cys481 and on the metabolic site guided by metabolic study were conducted. 7S was identified as the most potent showing an IC₅₀ value of 0.4 nM against BTK and 16 nM against BTK-dependent TMD8 cells. Compared to 1, 7S was slightly more selective with strong inhibition on the B-cell receptor signaling pathway. In a TMD8 cell-derived animal xenograft model, 7S showed a relative tumor volume of 5.3 at 15 mg/kg QD dosage that was more efficacious than 1 (RTV 6.6) at a higher dose of 25 mg/kg QD. All these results suggest 7S as a new BTK inhibitor worthy of further profiling.

Full text of DOI:10.1021/acs.jmedchem.8b00441

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Article
Discovery of 4,7-Diamino-5-(4-phenoxyphenyl)-6-methylene-pyrimido[5,4-
b]pyrrolizines as Novel Bruton’s Tyrosine Kinase (BTK) Inhibitors
Yu Xue, Peiran Song, Zilan Song, Aoli Wang, Linjiang Tong, Mei-
Yu Geng, Jian Ding, Qingsong Liu, Liping Sun, Hua Xie, and Ao Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00441 • Publication Date (Web): 01 May 2018
Downloaded from http://pubs.acs.org on May 1, 2018
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Discovery of
,7ꢀDiaminoꢀ5ꢀ(4ꢀphenoxyphenyl)ꢀ6ꢀmethyleneꢀpyrimido[5,4ꢀb]pyrro
lizines as Novel Bruton’s Tyrosine Kinase (BTK) Inhibitors
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†
,‡,ǁ
ǂ,ꢁ,ǁ
‡,ǁ
∫
ǂ
Yu Xue, Peiran Song, Zilan Song, Aoli Wang, Linjiang Tong, Meiyu
ǂ,§,ꢁ
ǂ,§,ꢁ
∫
†,*
ǂ,§,*
‡,§,ꢁ,*
Geng,
Jian Ding,
Qingsong Liu, Liping Sun, Hua Xie, Ao Zhang
†
‡
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
CAS Key Laboratory of Receptor Research, and the State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
ǂ
Division of Antiꢀtumor Pharmacology, the State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
∫
High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, China
§
College of Pharmacy, University of Chinese Academy of Sciences, China
ꢁ
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
ǁAll these authors contributed equally to this work.
∗
To whom correspondence should be addressed. For A.Z.: phone: +86ꢀ21ꢀ50806035;
fax: 86ꢀ21ꢀ50806035; Eꢀmail: aozhang@simm.ac.cn; For H.X.: phone:
+86ꢀ21ꢀ50805897; fax: 86ꢀ21ꢀ50805897; Eꢀmail: hxie@simm.ac.cn; For L.S.: phone:
+
86ꢀ25ꢀ83271414; fax: 86ꢀ25ꢀ83271414; Email: chslp@cpu.edu.cn
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Abstract:
An alternative medicinal chemistry approach was conducted on BTK inhibitor 1
ibrutinib) by merging the pyrazolo[3,4ꢀd]pyrimidine component into a tricyclic
(
skeleton. Two types of compounds were prepared, and their biochemical activities on
BTK as well as stereochemistry effects were determined. Structural optimization
focusing on the reactive binding group to BTK Cys481 and on the metabolic site
guided by metabolic study were conducted. 7S was identified as the most potent
showing an IC50 value of 0.4 nM against BTK and 16 nM against BTKꢀdependent
TMD8 cells. Compared to 1, 7S was slightly more selective with strong inhibition on
Bꢀcell receptor signaling pathway. In TMD8 cellꢀderived animal xenograft model, 7S
showed Relative Tumor Volume of 5.3 at 15 mg/kg QD dosage that was more
efficacious than 1 (RTV 6.6) at higher dose of 25 mg/kg QD. All these results suggest
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7S as a new BTK inhibitor worthy of further profiling.
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INTRODUCTION
The Bruton’s tyrosine kinase (BTK) protein is a nonreceptor tyrosine kinase
belonging to the Tec kinase family (the other members are BMX, ITK, TEC and
1
TXK). It plays a critical role in Bꢀcell receptor (BCR) signaling by mediating B cell
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development and functioning. Therefore, dysregulation of BTK generally causes
2
severe leukemias and Bꢀcell related lymphomas. BTK is the upstream activator of
multiple antiapoptotic signaling molecules and networks, and is primarily expressed
in hematopoietic cells, particularly in B cells, but not in T cells or normal plasma
3
cells. The essential role of BTK in BCR signaling pathway and its restricted
expression pattern warrant it as a viable and attractive therapeutic target for the
4
treatment of Bꢀcell malignancies. The firstꢀgeneration multiꢀtargeted BTK inhibitor
5
ibrutinib (1, PCIꢀ32765) (Figure 1A) has been approved by FDA in 2013 to treat
mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), and various
6
,7
clinical trials are ongoing for new indications. Compound 1 is an irreversible
inhibitor by covalently binding to Cys481 of BTK with its acrylamido moiety. In
addition, 1 also irreversibly binds to other kinases such as epidermal growth factor
receptor (EGFR), ITK and TEC which might be attributed to the clinically observed
adverse effects in ibrutinibꢀtreated patients, including rash, diarrhea, arthralgias, atrial
7
fibrillation, ecchymosis and major hemorrhage. Though it is not for sure the
offꢀtargets also contribute to the clinical antitumor efficacy of 1, the objective of the
secondꢀgeneration BTK inhibitors is to achieve better BTK onꢀtarget selectivity to
8
minimize the side effects. As shown in Figure 1, several compounds such as
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acalabrutinib (2, ACPꢀ196), tirabrutinib (3, ONO/GSꢀ4059) and spebrutinib (4,
CCꢀ292) are being extensively studied in the clinic both for evaluation of their
therapeutical efficacies and for their safety profiles. Very recently, compound 2
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possessing higher selectivity and inhibitory effect against BTK received the FDA’s
approval as a bestꢀinꢀclass treatment for CLL with a 100% response rate for patients
positive for the 17p13.1 gene deletion ꢀ a subgroup of patients that typically results in
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a poor response to therapy and low expected clinical outcomes. Intriguingly,
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compound 4, another secondꢀgeneration BTK inhibitor also irreversibly binding the
Cys481 of BTK in high selectivity shows a clinical activity (in particular, durability of
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response) inferior to that of 1 or 2.
In addition to covalent inhibitors, several
1
3ꢀ15
nonꢀcovalent BTK inhibitors have also been reported.
These nonꢀcovalent
inhibitors could not form irreversible binding to BTK Cys481 because of absence of
the reactive binding group. However, they could target the binding pocket of inactive
BTK conformations through conformational changes, which offers reversible
16
interaction and binding to BTK. Although the precise reason is unclear for the
different clinical outcomes of these compounds, more diverse inhibitors with both
17
novel structures and selective onꢀtarget binding profile are needed.
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A. Clinical BTK inhibitors bearing an acrylamido or butynamido moiety to covalently interact with Cys481
O
O
O
N
O
O
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NH
HN
NH2
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NH2
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NH2
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NH
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N
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H
O
1
(ibrutinib, PCI-32765) 2 (acalabrutinib, ACP-196) 3 (triabrutinib, ONO/GS-4059) 4 (spebrutinib, CC-292)
B. Design of our new BTK inhibitors I and II
O
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X
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NH2
ring-merging
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6
,7,8,9-tetrahydropyrimido
6-methylene-7,8-dihydro-6H-
pyrimido[5,4-b]pyrrolizine
1
[5,4-b]indolizine
Figure 1. Clinical BTK inhibitors 1ꢀ4 (A) and our design of new BTK inhibitors I and II (B).
Rather than replacing the central hinge binder pyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine
template of 1 with other bicyclic (e.g. imidazo[1,5ꢀa]pyrazinꢀ8ꢀamine in 2 and
6ꢀaminoꢀ7Hꢀpurinꢀ8(9H)ꢀone in 3) or monocyclic (e.g. 2,4ꢀdiaminopyrimidine in 4)
bioisosteres, we recently conducted a different medicinal chemistry approach by
merging the pyrazolo[3,4ꢀd]pyrimidine component and the piperidine ring of 1 into a
tricyclic skeleton I (Figure 1B). Interestingly, during the synthesis of I, we found that
a change of the cyclization reaction condition led to a different cyclization product II.
Both compound series I and II contain brand new core structures that are rarely seen
18
in other categories of therapeutical drugs. Herein, we report the design, synthesis,
and pharmacological evaluations of both series of compounds as novel BTK
inhibitors.
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Figure 2. Binding modes of compounds 1 (A), 5 (B) and 7 (C) with BTK (PDB id: 3GEN).
RESULTS AND DISCUSSION
StructureꢀBased Drug Design. From the covalent docking model of 1 to BTK
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(
PDB id: 3GEN), the acrylamido moiety covalently binds to the Cys481 in the
active site of BTK to achieve a potent and irreversible inhibition (Figure 2A). In
addition, the pyrazolo[3,4ꢀd]pyrimidine backbone of 1 forms three critical hydrogen
bonds with hinge residues Met477, Glu475 and Thr474, respectively, through an
edgeꢀtoꢀface πꢀπ interaction between the terminal phenyl group and Phe540 in the
hydrophobic pocket. The pyrazolopyrimidine backbone of 1 proves to be effective in
developing BTK inhibitors, and a dozen of mimetics (e.g. 2ꢀ4) of this bicyclic
20
framework have been reported. A close examination of the binding mode of 1 with
BTK reveals to us that there is a large space between the nearly orthogonal pyrazolyl
and piperidyl groups, indicating that occupying this space with an appropriate ring
might be well tolerated. Initially, we considered to directly cyclize the pyrazolyl and
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piperidyl moieties of 1 to form a tetracyclic artwork. However, consideration of the
potential ring tension and the synthetic difficulty of the proposed tetracyclic backbone,
a cutꢀoff of the piperidyl ring is necessary, thus leading to our first design of the
tricyclic compound series I (Figure 1B).
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Preliminary Evaluation of Compounds 5ꢀ7. The tricyclic compound 5 was
synthesized as a prototypic representative of compound series I. The tricycle
pyrimido[5,4ꢀb]pyrrolizine 7 (compound series II) was also obtained during
optimization of the cyclization reaction condition. A docking of 5 to BTK was shown
in Figure 2B, in which the critical irreversible binding, key hydrogen bonds and
hydrophobic interaction as that showed in 1 were all retained. Interestingly,
compound 7 bearing an exocyclic double bond also maintained the key interactions
with BTK (Figure 2C) and the exocyclic double bond had no significant contacts in
the catalytic domain. This analysis suggested that compounds 5 and 7 might be potent
BTK inhibitors.
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Figure 3. Characterizations of compounds 5ꢀ7 as potent BTK inhibitors. (A) Chemical structures
of 5ꢀ7. (B) In vitro kinase inhibitory activities of compounds against wildꢀtype BTK. (C) In vitro
C481S
kinase inhibitory activities of compounds 7 and 1 against BTK
mutant.
BTK inhibitory activity of compounds 5 and 7 were evaluated by using
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wellꢀestablished ELISA assays. The saturated compound 6 was prepared as a
comparison and compound 1 (ibrutinib) was included as a positive control (Figure
3A). As shown in Figure 3B, compounds 5 and 7 bearing an acrylamido moiety were
highly potent BTK inhibitors with IC50 values of 5.3 nM and 2.8 nM, respectively,
which were only 13ꢀfold and 7ꢀfold less potent than 1. Compound 6 bearing a
nonreactive propylamido moiety was approximately 1040ꢀfold less potent (IC50 of
417.4 nM) than 1. The significant discrepancy in biochemical activity between 5 and
1
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6 illustrated the necessity of the acrylamido warhead for irreversible covalent binding
C481S
with BTK Cys481. The effects of compound 7 against BTK
mutant were further
examined (Figure 3C). Compared to the high potency against wildꢀtype BTK,
C481S
compound 7 exhibited 165ꢀfold reduction of potency against BTK
(IC50 of 463.6
vs 2.8 nM). These results suggested that the new tricyclic compounds 5 and 7 were
potent BTK inhibitors and might covalently bind to the Cys481 leading to irreversible
5
inhibition on BTK kinase activity, a similar interaction profile to that of 1.
Kinase Selectivity Study of Compounds 5 and 7. To determine possible
offꢀtargets, we further analyzed the kinase selectivity of the two new BTK inhibitors 5
21
and 7 by the KINOMEScan™ screening platform. Both compounds were tested
against a panel of 468 kinases and mutants at 1 ꢂM concentration and the results were
showed in Figure 4. Compound 7 was found to possess a better selectivity (Sꢀscore (1)
= 0.007), whereas the S(1) value of 5 was 0.027. Not surprisingly, the major
offꢀtargets of both compounds were those kinases bearing a cysteine residue in the
ATP binding pocket similar to BTK, which could be covalently bound by the
acrylamido warhead, including BLK, EGFR, EebB4, TEC and so on. More
specifically, compound 7 had only three major offꢀtargets (BLk, ErbB4, MEK) with a
percent control number less than 1 at 1 ꢂM concentration, a selectivity profile much
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Number of
NonꢀMutant
Kinases
Selectivity
Compd.
Number of
Hits
Selectivity
Score
Score Type
S(35)
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0.117
0.074
0.027
0.082
0.047
0.007
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S(10)
S(1)
S(35)
S(10)
S(1)
Figure 4. Human kinome wide selectivity profiling of compounds 5 and 7 in DiscoveRx
KINOMEScan™ screening platform. Measurements were performed at 1 ꢂM concentration of the
compounds. Upper panel: TREEspot™ interaction maps for 5 and 7 in 468 kinase targets. Lower
panel: Sꢀscores of 5 and 7 with percent control numbers less than 35, 10 and 1, respectively.
Structure Optimization on Lead Compound 7. In view of the structural novelty,
high potency and better kinase selectivity profile, we selected compound 7 as our
tricyclic lead BTK inhibitor for further structural optimization. First, to identify the
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optimal covalent warhead binding to BTK Cys481 residue, a panel of reactive
acrylamido groups was tested (Table 1). First, we inserted variant amino acid moieties
into the acrylamido fragment and the corresponding compounds 8ꢀ11 showed reduced
potencies against BTK. Compared to compound 7, insertion of smaller amino acid
moieties such as 2ꢀaminoacetyl (8, IC50 of 23.6 nM), 2ꢀaminopropanoyl (9, IC50 of
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
40.1 nM) or 1ꢀaminocyclopropanecarbonyl (10, IC50 of 23.8 nM) led to 8ꢀ to 14ꢀfold
loss of potency, and insertion of a longer amino acid moiety such as
3ꢀaminopropanoyl (11, IC50 of 725.2 nM) significantly reduced the inhibitory potency.
Substitution on the terminal vinyl carbon with a dimethylaminomethyl yielded
compound 12, which retained high potency with an IC50 value of 4.3 nM. Notably,
replacing the acrylamido moiety in 7 with a butꢀ2ꢀynamido moiety provided
compound 13 showing an IC50 value of 2.1 nM, a potency even higher than that of 7
(IC50 of 2.8 nM). Compound 14 lacking the acryloyl warhead completely lost the
potency (IC50 > 1000 nM), whereas saturation of the acrylamido with nonreactive
propionyl moiety led to 22ꢀfold reduction of potency (15, IC50 of 62.6 nM). These
results further confirmed that the existence of a Michael acceptor warhead was
essential for BTK’s covalent binding, and the butꢀ2ꢀynamido (13, IC50 of 2.1 nM) was
slightly better than the prototypic acrylamido moiety (7, IC50 of 2.8 nM).
Table 1. The in vitro BTK inhibition of compounds bearing different covalent or noncovalent
reactive groups.
1
a
Compd.
R
BTK IC50 (nM)
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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
7
8
9
2.8 ± 1.1
23.6 ± 4.5
40.1 ± 4.7
23.8 ± 5.0
725.2 ± 182.5
4.3 ± 0.7
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
10
11
1
2
3
1
2.1 ± 0.2
14
> 1000
15
62.6 ± 13.4
a
IC50 values are reported as means of duplicates.
Chiral Resolution of Potent BTK inhibitors 5, 7 and 13. Since compound 1 is
an optically pure Rꢀenantiomer in the stereogenic carbon center, its Sꢀenantiomer as
5
a
well as the racemate are less potent. To determine where this was also the case in
our new tricyclic BTK inhibitors, the Rꢀ and Sꢀenantiomers of the three potent
racemic compounds 5, 7 and 13 were prepared and tested. As shown in Table 2,
enantiomers 5R and 5S showed nearly identical IC50 values (3.0 vs 2.8 nM).
Surprisingly, significant discrepancy was observed between the two enantiomers of 7
and 13 both bearing the pyrimido[5,4ꢀb]pyrrolizine framework. In both cases, the
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Sꢀenantiomers were more potent than their corresponding Rꢀenantiomers. Both 7S and
13S showed high potency with an identical IC50 value of 0.4 nM, which was equal to
that of 1 (IC50 of 0.4 nM), whereas the IC50 values of Rꢀenantiomers 7R and 13R were
6.1 and 3.3 nM, respectively. The similar biochemical activity of the enantiomers 5S
and 5R on BTK binding is likely ascribed to the fact that the newly formed piperidine
ring in 5 could torture to adapt a configuration change upon covalently binding to the
active site of BTK. However, the newly formed pyrrole ring in 7 and 13 have more
ring and steric strain that is disadvantageous for ring tortuosity.
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. The in vitro BTK inhibition of the stereoisomers of potent compounds 5, 7 and 13.
a
Compd.
Configuration
BTK IC50 (nM)
5
(racemate)
5.3 ± 0.2
5
R (Rꢀenantiomer)
3.0 ± 1.9
2.8 ± 0.3
2.8 ± 1.1
6.1 ± 1.4
0.4 ± 0.1
2.1 ± 0.2
3.3 ± 1.1
0.4 ± 0.1
5S (Sꢀenantiomer)
7 (racemate)
7
7
1
1
1
R (Rꢀenantiomer)
S (Sꢀenantiomer)
3 (racemate)
3R (Rꢀenantiomer)
3S (Sꢀenantiomer)
a
IC50 values are reported as means of duplicates.
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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
Pharmacokinetic (PK) Parameters of the Selected BTK Inhibitors 7S and
13S. Since both 7S and 13S showed high potencies against BTK kinase, they were
selected for pharmacokinetic study in rats following intravenous (1 mg/kg) and oral (3
mg/kg) administration. As a comparison, 1 was also tested in the same conditions. As
shown in Table 3, both 7S and 13S have similar halfꢀlives (T1/2 = 0.57 h and 0.54 h,
respectively), which were slightly longer than that of 1 (T1/2 = 0.42 h). Meanwhile,
better plasma exposure after oral administration was observed for both tricyclic
compounds, and the AUC0ꢀ∞ values for 7S and 13S were 4ꢀ and 10ꢀfold higher than
that of 1, respectively. The same trend was observed in their Cmax values. In addition,
both 7S and 13S showed lower clearances than 1. Finally, the oral bioavailabilities of
7S and 13S were calculated as 18.3% and 35.8%, respectively, which were much
higher than that of 1 (F = 4.00%).
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
Table 3. PK parameters of the new BTK inhibitors 7S and 13S in rats.
iv (1 mg/kg)
po (3 mg/kg)
Compd.
1
CL
L/h/Kg)
V
ss
T
(h)
1/2
C
max
T
(h)
max
AUC0ꢀ∞
(ng·h/mL)
F
(%)
(
(L/kg)
1838
1299
1390
(ng/mL)
61.2
46.3
32.8
0.42
0.57
0.54
39
0.25
0.25
0.25
57
4.00
18.3
35.8
7
S
223
211
557
13S
340
a
Values are the average of three determinations. Vehicle: DMSO, Tween 80, normal saline. CL,
clearance; Vss, volume of distribution; T1/2, halfꢀlife; Cmax, maximum concentration; Tmax, time of
maximum concentration; AUC0ꢀ∞, area under the plasma concentration time curve; F, oral
bioavailability.
Metabolic Stability Study of the Selected BTK Inhibitors 7S and 13S. To
evaluate the metabolic stability of the new tricyclic BTK inhibitors 7S and 13S, we
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investigated the potential metabolites of both compounds by incubating with liver
microsomes of different species (mouse, rat and human) for three hours. The major
metabolites were tested by LC/MS (supporting information, Table S1). Both
compounds showed moderate to good stability with similar metabolic products in the
liver microsomes. Compound 7S was more stable in mouse liver microsomes (MLM)
than in human liver microsomes (HLM) and rat liver microsomes (RLM). Differently,
compound 13S was much more stable in HLM and MLM, but not stable in RLM. In
general, the major metabolic pathway for both compounds was the paraꢀoxidation of
the phenyl group in the diphenyl ether component leading to paraꢀhydroxylated
compound Mꢀ1, accounting for 16.5% and 18.8%, respectively, of the total
metabolites (including parent compound Mꢀ0) in HLM (Table S1). In addition,
substantial dihydroxylation occurred in both the vinyl (7S) and ethynyl (13S) moieties
across the three liver microsomes. The proposed metabolic pathways of both
compounds were shown in Figures 5A and 5B.
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
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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
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4
5
5
5
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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. Metabolic study of compounds 7S and 13S in liver microsomes of different species.
Further Modification of Compound 7S. Since the metabolic stability study in
liver microsome revealed that the most active metabolic site of compound 7S might
be the paraꢀposition of the terminal phenyl group. Therefore, two approaches were
conducted to eliminate or ultimately reduce the paraꢀphenyl oxidative metabolism
(
Table 4). First, we introduced a small series of substituents to the paraꢀposition of the
terminal phenyl to block the hydroxylation liability. It was found that all the
substituents, including ꢀtꢀBu (16), ꢀF (17), ꢀCF (18) and ꢀOCF (19) groups, appended
3
3
in the paraꢀposition caused 5ꢀ to 19ꢀfold decrease of potency, comparing to that of 7S.
The reduced potency was likely due to the steric hindrance in the hydrophobic
binding pocket. In the meantime, replacement of the terminal phenoxy fragment with
various Nꢀaryl or Nꢀarylmethyl carbamic groups was conducted to block the active
metabolic site. To our delight, compared to 7S, compound 20 bearing a
pyridieꢀ2ꢀylcarbamic group showed compatible biochemical potency against BTK
with an IC50 value of 0.5 nM. Compound 21 bearing an additional trifluoromethyl
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group also retained high potency of 0.8 nM. Replacing the terminal phenoxy group of
7S with (1ꢀ(pyridinꢀ2ꢀyl)ethyl)carbamic moiety significantly reduced the potency (22,
IC50 of 50.4 nM), whereas slightly lower potency was observed for compounds 23 and
2
4, both bearing a benzylcarbamic moiety and showing IC values of 3.1 nM and 4.3
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
nM, respectively.
Table 4. The in vitro BTK inhibition of compounds modified in terminal phenyl to block the
paraꢀoxidative metabolism.
2
a
Compd.
7S
R
BTK IC50 (nM)
0.4 ± 0.1
1
6
7
3.7 ± 0.9
2.2 ± 1.5
7.4 ± 2.5
3.5 ± 1.7
0.5 ± 0.1
0.8 ± 0.1
1
18
1
9
0
2
21
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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
2
2
50.4 ± 9.4
3.1 ± 1.0
23
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
24
4.3 ± 1.7
a
IC50 values are reported as means of duplicates.
Antiproliferative Effects of Compounds 7S, 20 and 1. Since the optimized
compound 20 showed similar high potency against BTK as that of the lead compound
7S and the approved drug 1, both 7S and 20 were selected for antiproliferative effect
study in the Bꢀcell lymphoma (Ramos and TMD8) cells. Compound 1 was also tested
as a comparison. Both Ramos and TMD8 cells are Bꢀcell lymphoma cell lines
expressing BTK protein, but the Ramos cells are not strongly depended on it, whereas
4b
the survival of TMD8 cells is strongly dependent on the expression of BTK protein.
As shown in Table 5, compounds 7S and 20 suppressed the proliferation of Ramos
cells in micromolar range with IC50 values of 5.03 ꢂM and 14.3 ꢂM, respectively,
whereas the approved drug 1 showed an IC50 value of 0.92 ꢂM. This result confirmed
that 1 was a nonꢀselective BTK inhibitor and its antitumor activity was a consequence
of multiple kinase inhibition. The lower potency of the new inhibitors 7S and 20
indicated that these new tricyclic compounds were more BTKꢀselective. This analysis
was further confirmed by the high antiproliferative potency against the BTK highly
sensitive TMD8 cells. All the three compounds 1, 7S and 20 showed low nanomolar
potency with IC50 values of 10 nM, 16 nM and 4 nM, respectively in this cell line.
Table 5. Target inhibition and antiproliferative effects of selected compounds.
a
Compd.
IC50 (ꢂM)
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BTK
Ramos
0.92 ± 0.1
5.03 ± 0.85
14.3 ± 6.1
TMD8
1
0.0004 ± 0.0001
0.0004 ± 0.0001
0.0005 ± 0.0001
0.010 ± 0.004
0.016 ± 0.010
0.004 ± 0.003
7
S
0
2
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
IC50 values are reported as means of duplicates.
From the results above, both the new inhibitors 7S and 20 showed high potency
and selectivity against BTK, especially for compound 20. Unfortunately, further
pharmacokinetic study on 20 showed a lower oral bioavailability of 8.24%
(
Supporting Information, Table S2). Taking together, compound 7S showed overall
optimal drug candidacy and was elected for further profiling.
Covalent docking mode of 7S with BTK. Similar to binding mode of compound
7 with BTK in Figure 2C, the specific Sꢀenantiomer 7S could covalently bind to BTK
Cys481. As shown in Figure 6, 7S overlapped well with 1, including the bicyclic
skeleton as well as the hydrophobic moiety. The sulfhydryl of Cys481 rotated about
o
6
8 to adapt the newly formed covalent bond between the acrylamido moiety of 7S
and BTK Cys481. Critical hydrogen bonds with hinge residues Met477, Glu475 and
Thr474 were also observed with distances of 2.0, 1.9 and 2.8 Å, respectively. In
addition, there is a πꢀπ interaction between the terminal phenyl group and Phe540.
Figure 6. Covalent docking mode of compound 7S (green) with BTK (PDB id: 3GEN)
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8
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
overlapping with 1 (purple).
Kinase Selectivity Profile of 7S. On the basis of the KINOMEScan™ screening
results of the racemic compound 7 (Figure 4), we further tested the biochemical
activity of compound 7S against a panel of 14 kinases. Similar to 1, the major
offꢀtarget kinases of 7S were those with cysteine residue in the ATP binding pocket,
which could be irreversibly bound by the covalent reactive group (Table 6). As such,
compound 7S showed a high potency against EGFR (IC50 of 1.3 nM), but variant
potency against the other members of the Tec kinase family with IC50 values of 174.1,
15.1, 1.4 and 2.5 nM for BMX, ITK, TEC and TXK, respectively. Although the
overall selectivity was similar to 1, 7S showed greater BTKꢀselectivity than 1 against
BMX (174.1 vs 5.8 nM), RET (20.3 vs 5.2 nM) and ErbB2 (23.2 vs 1.5 nM). It was
not clear whether such difference in kinase selectivity between 7S and 1 was
beneficial to the antitumor efficacy or clinical safety, a careful balance of the clinical
outcomes should be taken if compound 7S finally proceeds to clinical trials.
Table 6. Kinase selectivity profile of compound 7S.
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
IC50 (nM)
Kinase
7
S
1
BTK
BLK
ErbB4
EGFR
TEC
0.4 ± 0.1
0.2 ± 0.1
0.6 ± 0.2
1.3 ± 0.3
1.4 ± 0.6
2.5 ± 0.5
15.1 ± 3.2
20.3 ± 6.6
23.2 ± 1.5
0.4 ± 0.2
0.5 ± 0.2
1.8 ± 1.0
1.2 ± 0.3
0.5 ± 0.3
3.0 ± 1.8
13.9 ± 1.6
5.2 ± 2.7
1.5 ± 0.9
TXK
ITK
RET
ErbB2
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Fltꢀ3
BMX
55.0 ± 11.7
174.1 ± 93.3
214.5 ± 126.0
> 1000
>1000
5.8 ± 2.3
> 1000
> 1000
> 1000
PDGFRꢀβ
EPHꢀA2
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
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6
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3
4
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6
7
8
9
0
CSF1R
> 1000
a
IC50 values are reported as means of duplicates.
Effects of Compound 7S on TMD8 cells. BTK plays an imperative role in BCR
signaling pathway, which is relevant to several severe leukemias and lymphomas.
BCR is first activated by CD79, which causes activation of upstream kinases, such as
SYK (Spleen tyrosine kinase). Then BTK is recruited to cell membrane and
phosphorylated in its SH2 domain. The activated BTK then phosphorylates its
substrate phospholipase γ2 (PLCγ2), leading to activation of the downstream
22
signaling pathways, such as NFꢀκB and STAT3. In the BTKꢀdependent diffuse large
Bꢀcell lymphoma (DLBCL) TMD8 cells, both compound 7S and 1 were found to
significantly inhibit the autophosphorylation of BTK at the Y223 site. Particularly, 7S
showed a doseꢀdependent inhibition with an IC50 value less than 10 nM. Furthermore,
7S inhibited downstream phosphorylation of Y759 of PLCγ2 comparable to that of 1
at 1 ꢂM (Figure 7A).
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9
0
Figure 7. Effects of compound 7S on TMD8 cells. (A) Inhibitory activity of 7S on BTK and
downstream signaling pathway in cells. Cells were starved in serumꢀfree medium before treated
with the indicated concentrations of compound 7S or 1 for 4 h and stimulated by antiꢀIGM. (B)
Washout experiment of 7S in TMD8 cells. (C) Effects of 7S on cell cycle in TMD8 cells. (D) 7S
induced apoptosis of TMD8 cells. Data are shown as meanꢃ±ꢃSD. Each experiment was conducted
*
**
independently for three times. Pꢃ<ꢃ0.05, Pꢃ<ꢃ0.01 vs control.
Meanwhile, we carried out a washout experiment to further confirm the
irreversibility of compounds 7S and 1 on the inhibition of BCR signaling pathway in
TMD8 cells (Figure 7B). After 2 h exposure of 7S and 1 at 1 ꢂM, the cells were
washed with PBS and collected in 0, 2, 4, 8, 12 and 24 h, the phosphorylated levels of
BTK at Y223 were detected. The results showed that 7S treatment for 2 h completely
inhibited the phosphorylation of BTK at Y223, and the inhibition could be observed
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even after 24 h washout. All these results suggested a strong irreversible inhibition of
7S on BTK, a profile similar to that of compound 1.
The effects of 7S on the cell cycle progression in TMD8 cells were investigated
as well. After 48 h treatment with TMD8 cells, 7S was found to significantly arrest
cell cycle progression at G1 phase in a doseꢀdependent manner (Figure 7C). Exposure
to 1 nM of 7S arrested 57% cells occupying the G1 phase, compared with 51% of
control cells. Approximately 77% of cells was arrested in G1 phase when the
concentration reached to 100 nM. We then proceeded to test the effects of 7S on
apoptosis in TMD8 cells. As illustrated in Figure 7D, 7S triggered
concentrationꢀdependent apoptosis of the cells with ratios of 40%, 49% and 79% at
concentrations of 10 nM, 100 nM and 1000 nM, respectively, compared with 11 % in
untreated control cells.
1
1
1
1
1
1
1
1
1
1
2
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8
9
0
Together, these results demonstrated that 7S, as a new inhibitor of BTK, induced
apoptosis and G1 phase cell cycle arrest, thus suppressing the growth of cancer cells.
Tumor Growth Inhibition of 7S in the TMD8 Xenograft Model. In view of the
promising in vitro profile of the new BTK inhibitor 7S, we then evaluated its
antitumor activity in vivo in female CBꢀ17 SCID nude mice bearing human TMD8
tumor cells. Both compounds 7S and 1 were administered orally (po) once daily (QD)
for 13 consecutive days till the vehicle group had to be euthanized due to the tumor
burden. As shown in Figure 8A, oral administration of 7S at 5 mg/kg showed minor
antitumor efficacy with a Relative Tumor Volume (RTV) of 11.1. However, at a
higher dose of 15 mg/kg QD, compound 7S significantly suppressed the tumor growth
with RTV of 5.3. The reference compound 1 showed a RTV of 6.6 at the dose of 25
mg/kg QD. The smaller RTV rate (5.3 vs 6.6) and lower dosage (15 vs 25 mg/kg) of
7S over 1 indicated that compound 7S had higher antitumor efficacy in vivo. Further,
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6
the significant suppression of both 7S and 1 on tumor growth was maintained to 17
days when the lower dosage group of 7S (5 mg/kg) had to be euthanized. In all four
groups, the mice slightly gained weight after treatment, and no significant weight
fluctuations were observed during the course (Figure 8B).
0
1
2
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8
9
0
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0
We also elucidated the effects of 7S on target inhibition in tumor by using
Western blot analysis. The data shows a significant reduction of pꢀBTK expression
upon Dꢀ39 treatment, accompanied with a decrease of pꢀPLCγ2 expression (Figure
2
3
8
C). The expression of Kiꢀ67, a proliferation marker in tumor, was evaluated by
using immunohistochemistry and H&E staining. A significant decrease of Kiꢀ67
expression was observed in 7S treatment group compared to the control group (Figure
8D). These results clearly demonstrated that 7S efficiently inhibited the activation and
downstream signaling transduction of BTK and thus suppressed the tumor growth.
Figure 8. In vivo TMD8 xenograft model study of compound 7S and 1. (A) The tumor growth
curve of four groups, including vehicle control, 1 (25 mg/kg, QD), 7S (5 mg/kg, QD) and 7S (15
mg/kg, QD). The vehicle group was euthanized on day 13 of treatment, and the other three groups
were executed on day 17 of treatment. (B) The body weights of the group mice over time. (C)
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Inhibition of target (BTK and PLCγ2) activation by 7S in tumors. (D) Immunohistochemical
∗
analysis of Kiꢀ67 in tumor tissues. P < 0.05 vs. control.
CHEMISTRY
Synthesis of Tricyclic Compounds. Pyrimido[5,4ꢀb]indolizine compounds 5 and
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
6
were prepared as shown in Scheme 1. The synthesis was started from the
Oꢀesterification of racemic Nꢀprotected alcohol 25 with methanesulfonyl chloride
MesCl) to generate the intermediate 26 in 85% yield. Substitution of
ꢀchloroꢀ5ꢀiodoꢀ7Hꢀpyrrolo[2,3ꢀd]pyrimidine with aminoalcohol 26 delivered
(
4
pyrrolo[2,3ꢀd]pyrimidine 27 in 74% yield. Ammonolysis of 27 with ammonium
hydroxide in a seal tube resulted in compound 28 in 75% yield. Pdꢀcatalyzed
SuzukiꢀMiyaura coupling of iodide 28 with 4ꢀphenoxyphenylboronic acid afforded
diphenyl ether 29 in 71% yield. Bromination of ether 29 with N-bromo succinimide
(NBS) afforded bromide 30 in 84% yield. Brown hydroboration of 30 in the terminal
double bond followed by intramolecular SuzukiꢀMiyaura coupling using PdCl dppf
2
catalyst delivered tricyclic compound 31 in 49% overall yield. NꢀDeprotection with
trifluoroacetic acid (TFA) followed by acylation with acrylic anhydride or carboxylic
acid afforded compound 5 or 6 in 58% and 73% yields, respectively.
a
Scheme 1. Synthesis of compounds 5 and 6.
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1
1
1
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2
2
2
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3
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5
5
5
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0
1
2
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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
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9
0
1
2
3
4
5
6
7
8
9
0
a
o
o
Reactions and conditions: (a) MesCl, Et
3
N, CH Cl
2 2
, 0 C to rt, 85%; (b) K
2
CO
3
, DMF, N
, 1,4ꢀdioxane/H
, 0 C to rt; 2) PdCl dppf,
N,
, rt, 73% (two steps).
2
, 55 C,
o
7
3
4%; (c) NH ·H
2
O, 1,4ꢀdioxane, 120 C, seal tube, 75%; (d) Pd(PPh
)
3 4
, Na
2
CO
3
2
O,
o
o
9
0 C, 71%; (e) NBS, DMF, rt, 84%; (f) 1) 9ꢀBBN, anhydrous THF, N
2
2
o
NaOH, THF/H
2
O, N
2
, 85 C, 49% (two steps); (g) TFA, CH
2 2
Cl , rt; (h) acrylic anhydride, Et
3
o
CH
Cl
2 2
, 0 C to rt, 58% (two steps); (i) propanoic acid, HATU, Et
3
N, CH Cl
2
2
It has to be mentioned that during the preparation of tricyclic compound 31, the
yield was highly dependent on the quality of boronation agent
ꢀborabicyclo[3.3.1]nonane (9ꢀBBN). Lower quality of 9ꢀBBN led to both the
9
tricyclic 31 and 33. After optimization of the coupling conditions, we found that
compound 33 could be conveniently prepared in 88% yield through direct
intramolecular Heck reaction of the precursor 30 under the catalysis of Pd(PPh
3 4
)
(
Scheme 2). After removal of the NꢀBoc protecting group of 33 with TFA, subsequent
acylation with an appropriate acid or anhydride afforded the corresponding
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acylamides 7ꢀ13 and 15 in 45ꢀ91% yields.
a
Scheme 2. Synthesis of compounds 7ꢀ15.
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
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4
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4
4
5
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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
o
Reactions and conditions: (a) Pd(PPh
3
)
4
, NaOH, THF/H
2
O, N
2
, 85 C, 88%; (b) TFA, CH
2
Cl
2
, rt,
o
4
9
8%; (c) acrylic anhydride, Et N, CH Cl , 0 C to rt, 45% (two steps); (d) R ꢀCOOH, HATU, Et N,
3
2
2
3
CH Cl , rt, 66ꢀ91% (two steps).
2
2
Synthesis of Chiral Compounds. The optically pure isomers of compounds 5, 7
and 13 were prepared from the Rꢀ or Sꢀconfigurated isomer of 25, which was obtained
24
by following a literature procedure. As shown in Scheme 3, phthalimide was treated
with racemic 2ꢀvinyloxirane using Pd (C H ) Cl and (S,S)ꢀDACHꢀnaphthyl Trost
2
3
5 2
2
ligand leading to the Rꢀconfigurated intermediate 34R in a highly regioꢀ and
enantioselective fashion (yield 92%, ee 98%). The absolute configuration of this
intermediate was confirmed by Xꢀray diffraction analysis (Supporting Information,
o
Figure S1, Table S3). Subsequent treatment with CH
3
NH
2
in open air in 60 C
followed by NꢀBoc protection delivered 25R, the key intermediate for the preparation
of optically pure 5R, 7R and 13R. Similarly, the same reaction sequence using
(R,R)ꢀDACHꢀnaphthyl Trost ligand afforded the Sꢀconfigured isomer 25S, the key
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2
2
2
2
2
2
2
3
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3
4
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4
4
4
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5
5
5
5
5
5
5
5
5
5
6
intermediate for 5S, 7S, 13S, and 16ꢀ24. Although we failed to obtain the Xꢀray
analysis of these final compounds, a key precursor 30S from 25S was successfully
crystallized and its Xꢀray analysis confirmed the absolute configuration (Supporting
Information, Figure S2, Table S4).
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
Scheme 3. Synthesis of the Rꢀ or Sꢀisomers of compounds 5, 7 and 13.
a
2 3 5 2 2
Reagents and conditions: (a) Pd (C H ) Cl , (S,S)ꢀDACHꢀnaphthyl Trost ligand for 34R and
(
R,R)ꢀDACHꢀnaphthyl Trost ligand for 34S, Na
2 3 2 2 2
CO , CH Cl , N , rt, yield 92%, ee 98%; (b) 1)
o
NH
2
CH
3
, open to air, 60 C; 2) Boc
2
O, NaHCO
3
, ethyl acetate, 88% (two steps).
The synthesis of the Sꢀconfigured compounds 16ꢀ24 was shown in Scheme 4.
SuzukiꢀMiyaura coupling of the Sꢀconfigured iodide 28S, which was prepared from
5S, with aryl boronates 35aꢀi under Pd(PPh ) catalysis provided compounds 36aꢀi
2
3
4
in 61ꢀ85% yields. Bromination of 36aꢀi followed by intermolecular Heck reaction
with Pd(PPh or PdCl dppf yielded tricyclic compounds 38aꢀi in 53ꢀ81% yields.
3
)
4
2
Removal of the NꢀBoc protecting group followed by acylation with acrylic anhydride
provided the corresponding compounds 16ꢀ24 in 45ꢀ79% overall yields.
a
Scheme 4. Synthesis of chiral compounds 16ꢀ24.
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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
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3
3
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3
3
3
3
4
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5
5
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0
1
2
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5
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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
o
Reactions and conditions: (a) Pd(PPh
DMF, rt, 70ꢀ77%; (c) Pd(PPh (for 16ꢀ19) or PdCl
3ꢀ81%; (d) TFA, CH Cl , rt; (e) acrylic anhydride, Et
)
3 4
, Na
2
CO
3
, 1,4ꢀdioxane/H
2
O, N
2
, 90 C, 61ꢀ85%; (b) NBS,
o
3
)
4
2
dppf (for 20ꢀ24), NaOH, THF/H
2
O, N
2
, 85 C,
o
5
2
2
3
N, CH
2
Cl
2
, 0 C to rt, 45ꢀ79% (two steps).
CONCLUSIONS
In summary, using a structureꢀbased drug design strategy, a new medicinal
chemistry approach was conducted on the BTK inhibitor 1 (ibrutinib) by merging the
pyrazolo[3,4ꢀd]pyrimidine component and the piperidine ring 1 into a tricyclic
skeleton. Two series of compounds were prepared, and their binding model,
biochemical activity as well as effects of the stereochemistry on BTK were
determined. Subsequent structural optimization both focusing on the reactive
acrylamido group as the covalent binder to the BTK Cys481 residue and on the
metabolic active site were conducted leading to the tricyclic compound 7S as the most
potent and orally available BTK inhibitor. Compound 7S has a biochemical IC value
50
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