Discovery and Evaluation of Pyrazolo[3,4-d]pyridazinone as a Potent and Orally Active Irreversible BTK Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.9b00395

The identification and lead optimization of a series of pyrazolo[3,4-d]pyridazinone derivatives are described as a novel class of potent irreversible BTK inhibitors, resulting in the discovery of compound 8. Compound 8 exhibited high potency against BTK kinase and acceptable PK profile. Furthermore, compound 8 demonstrated significant in vivo efficacy in a mouse-collagen-induced arthritis (CIA) model.

Full text of DOI:10.1021/acsmedchemlett.9b00395

Subscriber access provided by BIU Pharmacie | Faculté de Pharmacie, Université Paris V
Letter
Discovery and Evaluation of Pyrazolo[3,4-d]pyridazinone
as a Potent and Orally Active Irreversible BTK Inhibitor
Xuejun Zhang, Xijun Sheng, Jie Shen, Shoubo Zhang, Wenjie Sun, Chunli Shen, Yi Li,
Jun Wang, Huqiang Lv, Minghui Cui, Yuchuan Zhu, Lei Huang, Dongling Hao, Zhibo
Qi, Guanglong Sun, Weifeng Mao, Yan Pan, Liang Shen, Xin Li, Guoping Hu, Zhen
Gong, Shuhua Han, Jian Li, Shuhui Chen, Ronghua Tu, Xuehai Wang, and Chengde Wu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00395 • Publication Date (Web): 11 Dec 2019
Downloaded from pubs.acs.org on December 11, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Discovery and Evaluation of Pyrazolo[3,4-d]pyridazinone as a Potent
and Orally Active Irreversible BTK Inhibitor
Xuejun Zhanga,b*, Xijun Shenga,b, Jie Shena,b, Shoubo Zhanga,b, Wenjie Suna,b, Chunli Shenc*, Yi Lic,
Jun Wangc, Huqiang Lvc, Minghui Cuic, Yuchuan Zhuc, Lei Huangc, Dongling Haoc, Zhibo Qic,
Guanglong Sunc, Weifeng Maoc, Yan Panc, Liang Shenc, Xin Lic, Guoping Huc, Zhen Gongc, Shuhua
Hanc, Jian Lic, Shuhui Chenc, Ronghua Tua,b, Xuehai Wangb, and Chengde Wuc
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aHubei Bio-pharmaceutical Industrial Technological Institute Inc, No. 666 High Tech Avenue, East Lake High Tech
Development Zone, Wuhan Hubei 430075, China
bHumanwell Healthcare (Group) Co., Ltd, No. 666 High Tech Avenue, East Lake High Tech Development Zone, Wuhan Hubei
430075, China
cDomestic Discovery Service Unit, WuXi AppTec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131,
China
KEYWORDS: BTK inhibitor, irreversible, pyridazinone, enzymatic potency, rheumatoid arthritis.
ABSTRACT: The identification and lead optimization of a series of pyrazolo[3,4-d]pyridazinone derivatives are described as a novel
class of potent irreversible BTK inhibitors, resulting in the discovery of compound 8. Compound 8 exhibited high potency against
BTK kinase and acceptable PK profile. Furthermore, compound 8 demonstrated significant in vivo efficacy in a mouse collagen-
induced arthritis (CIA) model.
Bruton’s tyrosine kinase (BTK) is an important member of
N
O
O
O
O
N
O
the Tec family of non-receptor tyrosine kinases mostly
expressed in B cells, mast cells and macrophages, and plays a
central role in B-cell survival, activation, proliferation,
differentiation and maturation through multiple signaling
pathways downstream of the B cell receptor (BCR) and Fcγ
receptor (FcR).1-4 Therefore, BTK has been considered as a
promising and attractive target for the treatment of B cell related
diseases, such as hematological malignancies and rheumatoid
arthritis (RA).5-6
HN
HN
NH2
N
NH2
N
N
N
H
N
N
N
N
H2N
N
N
N
N
O
O
O
N
N
N
F
S
O
O
N
N
N
N
H
N
H
O
O
O
HM71224
ONO-4029
CC-292
Evobrutinib
Ibrutinib
first-in-class irreversible BTK inhibitor for the treatment of
mantle cell lymphoma (MCL), chronic lymphocytic leukemia
(CLL) and Waldenström’s macroglobulinemia (WM).13-17
However, the indication development of RA was limited due to
possible off-target side effects.18 Subsequently, several other
irreversible BTK inhibitors,19-23 such as HM71224,19 ONO-
4029, 20 CC-292,21 and Evobrutinib,23 have been also advanced
into clinical trials for the treatment of autoimmune diseases
(Figure 1).
The above BTK inhibitors contain different pyrimidine
scaffolds with an acrylamide group as Michael acceptor in the
structures, which can form a covalent bond with Cys481.
Inspired by these pioneering works, our medicinal strategy was
to keep the acrylamide moiety as a Michael acceptor unchanged
initially, and search for a novel structural motif as the central
core, hoping to generate irreversible BTK inhibitor with potent
activity, improved PK property and safety. Herein we describe
the discovery of a novel 4-amino-1H-pyrazolo[3,4-d]pyridazin-
7(6H)-one scaffold and our early lead optimization efforts in
this series, resulting in compound 8 as a highly potent BTK
inhibitor for RA (Figure 2).24
In fact, many research groups have been focusing their efforts
on the development of small molecular BTK inhibitors. Based
on the binding modes with the BTK catalytic domains, the
reported small molecular BTK inhibitors are generally divided
into irreversible inhibitors and reversible inhibitors.7-12 The
irreversible BTK inhibitors can achieve a strong binding to the
relatively unique Cys481 residue of BTK enzyme and exhibit
powerful advantage.5 Among them, Ibrutinib is the most
successful example and has been approved by the US FDA as
the
Figure 1. Representative Chemical Structures of Irreversible
BTK Inhibitors
Figure 2. The Structure of Compound 8
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
calculated strain energy of its binding mode is high. Therefore,
Page 2 of 7
O
1
2
3
4
originally it was not recommended with high priority for the
synthesis. However, the high potency of compound 8 reflected
that it was sometimes tricky to evaluate compounds by using
ligand strain energy.
NH2
N
N
HN
N
O
5
6
7
8
N
Interestingly, the originally predicted binding modes
indicated that compounds 5 and 6 could covalently bind well on
3PIY,26 while both compounds showed poor potency. The
biological data are not consistent with the computational
modeling results. The reason was not clear until the co-crystal
structure of Ibrutinib (5P9J) was released in 2017.27
Compounds 5 and 6 could not bind well on 5P9J, which has a
typical DFG-in conformation, while 3PIY has an atypical DFG
conformation. In 5P9J, the Asp539 formed salt bridge with
Lys430, which narrowed down the binding pocket. Therefore,
the tricycles core of both compounds could not be
accommodated in the binding pocket. The docking template
with atypical DFG conformation seemed not appropriate for the
prediction in this case. Different from compounds 5 and 6, the
core structure of compound 8 is bicycle and comparably small.
Compound 8 could bind well on both typical DFG-in 5P9J and
atypical DFG-in 3PIY with high docking score (-11.66 and -
10.40 kcal/mol). In atypical DFG-in 3PIY, Phe540 is out of
typical DFG position. The terminal phenyl group of compound
8 inserted into the hydrophobic pocket, but could not form π-π
stacking with Phe540, which led to the decreased docking score
(-10.40 vs. -11.66 kcal/mol).
O
8
Our primary optimization is focused on probing different
scaffolds that replace a pyrazolo[3,4-d]pyrimidine in Ibrutinib
structure (Figure 3). Compound 1 was initially synthesized by
cleaving the pyrazolo ring of Ibrutinib and evaluated in vitro
activity against the BTK enzyme. Unfortunately, compound 1
displayed weak activity against BTK with IC50 values of 921
nM. Cyclization of compound 1 led to different products 2-4,
which also exhibited poor inhibition of the enzyme (>10 μM,
1675 nM, and >10 μM respectively). In addition, the
introduction of a phenyl group into the pyrazolo[3,4-
d]pyrimidine showed similarly poor potency to inhibit the BTK
kinase, possessing IC50 value of 1244 nM (5). Changing the
pyrimidinamine of compound 5 into isoxazol-3-amine resulted
in a 3.7-fold decrease in activity (6, IC50 = 4664 nM). The
replacement of pyrazolo[3,4-d]pyrimidine of Ibrutinib with
pyrazolo[3,4-d]oxazinone also afforded weaker potency against
BTK (7). To our delight, further replacing the “O” of the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pyrazolo[3,4-d]oxazinone ring with
pyrazolo[3,4-d]pyridazinone derivative 8, showing
significantly increased enzymatic activity with IC50 of 2.1 nM.
a
“NH” led to
a
Figure 4. Predicted Binding Mode of Compound 8 (a) and
Overlaid Compound 8 with Ibrutinib (b) on BTK (pdb: 5P9J)
Figure 3. Study on Novel Core and BTKa IC50
O
O
O
O
O
NH2
HN
HN
NH2
N
N
N
N
N
N
O
N
O
NH
N
NH
N
NH
NH
N
N
N
N
N
O
O
O
O
1
3
2
4
BTK IC50 > 10000 nM
BTK IC50 = 921 nM
BTK IC50 = 1675 nM
BTK IC50 > 10000 nM
O
O
O
O
NH2
NH2
NH2
H2N
N
N
N
N
N
N
N
N
O
N
HN
N
N
O
N
N
O
O
N
N
N
N
O
O
O
O
6
5
7
8
BTK IC50 = 4664 nM
BTK IC50 = 1244 nM
BTK IC50 > 1000 nM
BTK IC50 = 2.1 nM
aBTKIC50 values are shown as single determinations. BTK IC50 of Ibrutinib
is 2.0 nM.
Docking study of compound 8 with BTK was carried out via
covalent docking protocol of Glide.25 As illustrated in Figure 4,
compound 8 could covalently bind to Cys481 and formed HB
network with hinge key residues Met477, Glu475 and
gatekeeper Thr474 (Figure 4a). The binding mode of compound
8 could overlay well with that of Ibrutinib (co-crystalized with
BTK, pdb: 5P9J) (Figure 4b). The piperidine and diphenyl ether
groups of compound 8 formed the same orientation with that of
Ibrutinib, and the terminal phenyl group inserted into a
hydrophobic pocket to form π-π stacking with Phe540. Despite
compound 8 potentially can well bind to BTK model, the
Encouraged by this result, we commenced with investigation
on N-substituent groups in pyridazinone ring (R1) and
acrylamide moiety containing different alkyl rings (R2) to
obtain more potent irreversible BTK inhibitors. The biological
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
1
and pharmacokinetics data for compounds 8-19 was
summarized in Table 1. Replacing the proton of the
pyridazinone ring with a methyl led to decrease of potency for
compound 9 (2.1 vs 160 nM). Introduction of larger substituent
groups, such as -Et (10), -CH2CF3 (11), led to complete loss of
activity. This indicated that an extra hydrogen bond interaction
of NH in the pyridazinone ring with the carbonyl group of
Met477 played a critical role for maintaining BTK inhibitory
activity. We next turned to optimize the Michael acceptor
moiety of the molecule. Firstly, ring size of the cyclic amino
group was investigated. When piperidine in compound 8 was
replaced by pyrrolidine, the resulting compound 12 was also
very active (IC50 = 1.4 nM). However, this change reduced oral
Cmax (252 vs 137 nM) and exposure (128 vs 68 ng·h/mL).
Changing the size of ring into four-membered (13) or seven-
membered (14) ring resulted in 3- to 5-fold decrease in activity
(IC50 = 9.8 nM and 6.7 nM). With regard to compound 15,
containing an aza-spiroheptane framework as acrylamide
moiety, it exhibited a decreased BTK enzymatic potency, with
an IC50 value of 46 nM. In addition, the enzymatic activity of
compound 16 bearing acrylamide moiety at the para-position in
cyclohexane group was similar to that of compound 8 (1.6 vs
2.1 nM). Compound 16 had better Cmax and AUC suggesting
better PK profile than compound 8, however, a poor solubility
affected its druggability, and compound 16 demonstrated a
higher human plasma protein binding rate (see Supporting
Information S41). Moving the para-position acrylamide group
of compound 16 to meta-position, the resulting compound 17
showed 6.3-fold reduced BTK activity. The combination of a
short carbon linker and N-methyl acrylamide (18) appeared to
be beneficial in inhibiting the BTK (IC50 = 1.2 nM) enzyme,
albeit with a lower Cmax (123 vs 252 nM) and oral AUC
exposure (49.5 vs 128 ng·h/mL). An attempt to introduce
dimethyl aminomethyl group at the alkene terminus of
compound 18 led to decreased potency, with an IC50 value of 35
nM (19).
O
O
12
13
H
H
1.4
9.8
137
-
68
-
N
N
1
2
3
4
5
6
7
8
1
1
O
14
H
6.7
-
-
N
O
1
N
15
16
H
H
46
-
-
1
9
O
1.6
290
161
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
1
NH
17
H
10
-
-
O
O
1
18
19
H
H
12
35
123
-
49.5
-
N
O
1
N
N
aIC50 values are shown as single determinations. bOral Cmax (maximum
concentration) after 2 mg/kg oral dosing in male Balb/C mice. cOral AUC
(area under the curve) after 2 mg/kg oral dosing in male Balb/C mice.
Based on the structure of compound 8, we conducted further
SAR studies on the Michael acceptor moiety of this compound.
As shown in Table 2, we examined the effect of α, β-unsaturated
amide groups on potency. Replacing the acrylamide with vinyl
sulfonamide resulted in compound 20, which displayed a 2-fold
decrease in activity against BTK (IC50 = 4.2 nM). When a
dimethyl aminomethyl group was introduced at the terminal
alkene of acrylamide of compound 8, the potency of the
resulting compound 21 was similar to that of compound 8 (IC50
= 2.6 nM). However, a larger morpholine group attached to the
same position (22) showed a decreased activity relative to
compound 21 (13 vs 2.6 nM). In addition, other analogues
containing cyano cyclopropyl acrylamide (23) or 2-butynamide
(24) showed IC50 values of 22 nM and 12 nM, respectively.
Table 1.Structure-Activity Relationship with R1 and R2 Groups
Table 2. Structure-Activity Relationship with R3 Group
O
NH2
O
N
N
N
NH2
N
R1
O
R2
N
N
HN
N
Oral
AUCc
(ng.h/
mL)
O
BTKa
Oral
Cmax
(nM)
R3
N
b
Cpd
R1
R2
IC50
(nM)
BTKa
Cpd
20
R3
1
1
1
1
IC50 (nM)
O
O
O
O
8
9
H
Me
Et
2.1
252
128
N
N
N
N
1
S
O O
4.2
1
160
-
-
-
-
-
-
N
21
2.6
13
O
1
N
10
11
>1000
>1000
22
O
O
CN
CH2
CF3
1
23
22
O
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
Me
AUC0-last
(ng.h/mL)
604
642
643
318
1
1
2
24
12
O
F%
-
23.7
PO
-
10.3
PO
aIC50 values are shown as single determinations.
3
Mice
IV
IV
4
5
6
7
8
9
After this preliminary optimization of enzymatic activities,
we next examined in vitro and in vivo pharmacokinetic (PK)
profiles of compound 8. For comparison, the approved drug
Ibrutinib was also evaluated as reference. As presented in Table
3, compound 8 and Ibrutinib exhibited equivalent potency
against BTK kinase (2.1 nM vs 2.0 nM). They also showed
>95% plasma protein binding across 3 species of human, rat and
mouse. No major drug-drug interaction liability was detected
since inhibition against all 5 CYP enzyme was IC50 of >10 μM
except for Ibrutinib’s IC50 of 9.7 μM against 2C9. Similar hERG
liability was observed in compound 8 and Ibrutinib (5.6 vs 7.7
μM).
Dose
(mg/kg)
1
2
-
1
2
-
T1/2 (h)
0.42
31.3
0.82
252
0.34
27.4
0.55
55.3
Cl
-
-
(mL/min/kg)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Vdss(L/kg)
-
-
Cmax
(ng/mL)
-
-
AUC0-last
(ng.h/mL)
576
-
128
628
-
21.4
1.8
F%
11.2
Table 3. In Vitro Profiling Data for Compound 8 and Ibrutinib
Based on its enzymatic potency and pharmacokinetic profile,
compound 8 was taken forward for in vivo efficacy study in a
mouse collagen-induced arthritis (CIA) model.28 In this study,
compound 8 (3 mg/kg or 10 mg/kg) or Ibrutinib (10 mg/kg) was
dosed by oral gavage, q.d., from day 28 (10 mice per group). As
shown in Figure 5, the treatment with compound 8 or Ibrutinib
for 14 days inhibited the significant progression of the disease
compared to the vehicle control, and compound 8 exhibited a
clear dose-dependent reduction per paw clinical scores.
Notably, the 3 mg/kg regimen of compound 8 provided
equivalent efficacy to Ibrutinib at 10 mg/kg, and the best in vivo
efficacy was observed when dosed 10 mg/kg of compound 8
(Figure 5). Furthermore, compound 8 was also well tolerated,
and no significant body weight loss was observed for all
different dosages.
Cpd
8
Ibrutinib
Enzyme_IC50 (nM)
2.1
2.0
PPB %bound (h, r,
m)
97.5, 99.7, 95.3
96.9, 97.8, 96.2
1A2
>100
39.6
43.9
>100
37.3
5.6
>100
9.69
33.5
>100
40.8
7.7
2C9
CYP
2C19
2D6
3A4
inhibition
(μM)
hERG IC50 (μM)
Pharmacokinetic parameters of compound 8 and Ibrutinib in
the rat and mice were summarized in Table 4. After an
intravenous injection, similar pharmacokinetic properties with
half-life (rat: 0.32 vs 0.32 hour; mice: 0.42 vs 0.34 hour),
clearance (rat: 54.6 vs 53.2 mL/min/kg; mice: 31.3 vs 27.4
mL/min/kg), volume of distribution (rat: 1.55 vs 1.44 L/kg;
mice: 0.82 vs 0.55 L/kg) and AUC exposure (rat: 604 vs 643
ng.h/mL; mice: 576 vs 628 ng.h/mL) were observed in two
species. After oral administration, compound 8 exhibited higher
Cmax (rat: 466 vs 150 ng/mL; mice: 252 vs 55.3 ng/mL) and
plasma exposure (rat: 642 vs 318 ng.h/mL; mice: 128 vs 21.4
ng.h/mL) with a favorable oral bioavailability (rat: 23.7% vs
10.3%; mice: 11.2% vs 1.8%) compared to Ibrutinib.
Figure 5. In Vivo Efficacy of Compound 8 and Ibrutinib in a
Mouse Collagen-induced Arthritis (CIA) Model
Clinical score
7.5
Normal
6.0
Vehicle
Dosing
Compound 8 3 mpk
Compound 8 10 mpk
Ibrutinib 10 mpk
4.5
3.0
1.5
0.0
** ***
**
*** ***
***
***
***
***
***
***
**
***
***
***
***
20 22 24 26 28 30 32 34 36 38 40 42
Day
Table 4. Pharmacokinetic Parameters of Compound 8 and
Ibrutinib
Body weight
27
26
25
24
23
22
21
Cpd
8
Ibrutinib
IV
Rat
IV
2
PO
10
-
PO
10
-
Dose
(mg/kg)
2
T1/2 (h)
0.32
54.6
1.55
466
0.32
53.2
1.44
150
Cl
20 22 24 26 28 30 32 34 36 38 40 42
Day
-
-
(mL/min/kg)
Vdss(L/kg)
-
-
Scheme 1. The Synthetic Route of Compound 8
Cmax
(ng/mL)
-
-
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
O
O
Experimental and characterization data for all compounds and all
OEt
O
O
(i)
(iii)
(ii)
O
PhO
F
1
2
biological data. The Supporting Information is available free of
charge on the ACS Publications website.
O
PhO
25
26
27
3
4
5
AUTHOR INFORMATION
Corresponding Authors
O
O
(v)
(iv)
NC
I
6
7
*E-mail: zhangxuejun@renfu.com.cn;
*E-mail: shen_chunli@wuxiapptec.com
EtO
N
EtO
N
EtO
N
N
H
N
N
H
H
30
29
28
O
O
O
8
9
Author Contributions
O
O
OH
The manuscript was written by LY. All authors have given
approval to the final version of the manuscript.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NH2
NC
O
NBoc
(vii)
N
EtO
N
N
HN
N
(vi)
N
ABBREVIATIONS
32
31
O
PK, pharmacokinetics; SAR, structure-activity relationship; PPB,
plasma protein binding; CYP, cytochrome P450; hERG, human
ether-a-go-go related gene; IV, intravenous; PO, per oral; AUC,
area under curve; mpk, mg/kg.
NBoc
NBoc
O
O
O
NH2
NH2
OH
REFERENCES
(viii)
N
N
N
N
(x)
HN
HN
N
N
1. Vetrie, D.; Vorechovsky, I.; Sideras, P.; Holland, J.; Davies, A.;
Flinter, F.; Hammarstrom, L.; Kinnon, C.; Levinsky, R.; Bobrow, M.;
Smith, C. I.; Bentley, D. R. The gene involved in X-linked
agammaglobulinaemia is a member of the src family of protein-
tyrosine kinases. Nature 1993, 361, 226-233.
2. Tsukada, S.; Saffran, D. C.; Rawlings, D. J.; Parolini, O.; Allen,
R. C.; Klisak, I.; Sparkes, R. S.; Kubagawa, H.; Mohandas, T.; Quan,
S.; Belmont, J. W.; Cooper, M. D.; Conley, M. E.; Witte, O. N.
Deficient expression of a B cell cytoplasmic tyrosine kinase in human
X-linked agammaglobulinemia. Cell 1993, 72, 279-290.
3. Satterthwaite, A. B.; Witte, O. N. The role of Bruton's tyrosine
kinase in B-cell development and function: a genetic perspective.
Immunol. Rev. 2000, 175, 120-127.
4. Buggy. J. J.; Elias, L. Bruton tyrosine kinase (BTK) and its role
in B-cell malignancy. Int. Rev. Immunol. 2012, 31, 119-132.
5. Lou, Y.; Owens, T. D.; Kuglstatter, A.; Kondru, R. K.; Goldstein,
D. M. Bruton’s tyrosine kinase inhibitors: approaches to potent and
selective inhibition, preclinical and clinical evaluation for
inflammatory diseases and B cell malignancies. J. Med. Chem. 2012,
55, 4539-4550.
O
O
NH
N
O
33
8
Scheme 1
. (i) PhOH, t-BuOK; (ii) diethyl oxalate, NaH, toluene; (iii)
hydrazine hydrate, AcOH, EtOH; (iv) NIS, CAN, CH3CN; (v) CuCN,
Pd(dppf)Cl2, Pd2(dba)3, DMF; (vi) PPh3, DIAD, THF; (vii) hydrazine hydrate;
(viii) HCl/EtOAc; (x) HATU,DIPEA, DCM.
The compound 8, which is exemplified in the present article,
was prepared starting from the commercially available
materials, as outlined in Scheme 1. Compound 25 as the starting
material was treated by a series of reactions, including
substitution, elimination and cyclization, to provide the key
intermediate 28. The treatment of compound 28 with NIS in the
presence of CAN gave the corresponding product 29.
Compound 29 was reacted with CuCN in the presence of
o
Pd2(dba)3 and Pd(dppf)2Cl2 in DMF at 100 C to afford the
desired product 30. The treatment of compound 30 with (S)-N-
Boc-piperidin-3-ol under Mitsunobu reaction conditions
generated the product 31, followed by cyclization in refluxing
hydrazine hydrate leading to compound 32. Boc de-protection
of compound 32 with hydrochloric acid in EtOAc afforded
compound 33. Finally, the product 8 was obtained by the
condensation reaction of compound 33 with acrylic acid. This
synthetic sequence can be applied to the synthesis of
compounds 9-24 by varying the corresponding materials under
similar reaction conditions (Details for the synthesis of
compounds 1-7 and 9-24, see Supporting Information).
6. Hendriks, R. W.; Yuvaraj, S.; Kil, L. P. Targeting Bruton’s
tyrosine kinase in B cell malignancies. Nat. Rev. Cancer 2014, 14, 219-
232.
7. Akinleye, A.; Chen, Y.; Mukhi, N.; Song, Y.; Liu, D. Ibrutinib
and novel BTK inhibitors in clinical development. J. Hematol. Oncol.
2013, 6, 59-67.
8. Yao, X.; Sun, X. Y.; Jin, S. Y.; Yang, L.; Xu, H. J.; Rao, Y.
Discovery of 4-Aminoquinoline-3-carboxamide Derivatives as Potent
Reversible Bruton’s Tyrosine Kinase Inhibitors for the Treatment of
Rheumatoid Arthritis. J. Med. Chem. 2019, 62, 6561-6574.
9. Crawford, J. J.; Johnson, A. R.; Misner, D. L.; Belmont, L. D.;
Castanedo, G.; Choy, R.; Coraggio, M.; Dong, L. M.; Eigenbrot, C.;
Erickson, R.; Ghilardi, N.; Hau, J.; Katewa, A.; Kohli, P. B.; Lee, W.;
Lubach, J. W.; McKenzie, B. S.; Ortwine, D. F.; Schutt, L.; Tay, S.;
Wei, B. Q.; Reif, K.; Liu, L. C.; Wong, H.; Young, W. B. Discovery of
GDC-0853: A Potent, Selective, and Noncovalent Bruton’s Tyrosine
Kinase Inhibitor in Early Clinical Development. J. Med. Chem. 2018,
61, 2227-2245.
10. Liu, J.; Guiadeen, D.; Krikorian, A.; Gao, X. L.; Wang, J.; Boga,
S. B.; Alhassan, A. B.; Yu, Y. N.; Vaccaro, H.; Liu, S. L.; Yang, C. D.;
Wu, H.; Cooper, A.; Man, J. D.; Kaptein, A.; Maloney, K.; Hornak, V.;
Gao, Y. D.; Fischmann, T. O.; Raaijmakers, H.; Pham, D. V.; Presland,
J.; Mansueto, M.; Xu, Z. W.; Leccese, E.; Hoover, J. Z.; Knemeyer, I.;
Garlisi, C. G.; Bays, N.; Stivers, P.; Brandish, P. E.; Hicks, A.; Kim,
R.; Kozlowski, J. A. Discovery of 8-amino-imidazo[1,5-a]pyrazines as
reversible BTK inhibitors for the treatment of rheumatoid arthritis. ACS
Med. Chem. Lett. 2016, 7, 198-203.
In summary, we designed and synthesized a new series of
pyrazolo[3,4-d]pyridazinone derivatives as potent and orally
active irreversible BTK inhibitors. Through the exploration of
different N-substituent groups in pyridazinone ring and Michael
acceptor moieties in pyrazolo[3,4-d]pyridazinone, we
successfully developed the SAR profile around this series,
resulting in the discovery of compound 8, which has shown high
potency against BTK enzyme and acceptable oral PK.
Furthermore, compound 8 demonstrated prominent inhibition
of arthritis in a mouse collagen-induced arthritis (CIA) model.
ASSOCIATED CONTENT
Supporting Information
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
11. Lou, Y.; Han, X. C.; Kuglstatter, A.; Kondru, R. K.; Sweeney,
Hillmen, P.; Stephens, D. M.; Ghia, P.; Barrientos, J. C.; Pagel, J. M.;
Woyach, J.; Johnson, D.; Huang, J.; Wang, X.; Kaptein, A.; Lannutti,
B. J.; Covey, T.; Fardis, M.; McGreivy, J.; Hamdy, A.; Rothbaum, W.;
Izumi, R.; Diacovo, T. G.; Johnson, A. J.; Furman, R. R. Acalabrutinib
(ACP-196) in Relapsed Chronic Lymphocytic Leukemia. N. Engl. J.
Med. 2016, 374, 323-332.
23. Haselmayer, P.; Camps, M.; Bujalski, L. L.; Nguyen, N.;
Morandi, F.; Head, J.; O’Mahony, A.; Zimmerli, S. C.; Bruns, L.;
Bender, A. T.; Schroeder, P.; Grenningloh, R. Efficacy and
Pharmacodynamic Modeling of the BTK Inhibitor Evobrutinib in
Autoimmune Disease Models. J. Immunol. 2019, 202, 2888-2906.
24. Wang, X. H.; Wu, C. D.; Xu, Y.; Shen, C. L.; Li, L. E.; Hu, G.
P.; Yue, Y.; Li, J.; G, D. L.; Shi, N. Y.; Huang, L.; Chen, S. H.; Tu, R.
H.; Yang, Z. W.; Zhang, X. W.; Xiao, Q.; Tian, H.; Yu, Y. P.; Chen, H.
L.; Sun, W. J.; He, Z. Y.; Shen, J.; Yang, J.; Tang, J.; Zhou, W.; Yu, J.;
Zhang, Y.; Liu, Q. BTK Inhibitor. WO 2016112637 A1, July 21, 2016.
25. Schrödinger Release 2016-3: Glide, Schrödinger, LLC, New
York, NY, 2016.
26. Kuglstatter, A.; Wong, A.; Tsing, S.; Lee, S. W.; Lou, Y.;
Villasenñor, A. G.; Bradshaw, J. M.; Shaw, D.; Barnett, J. W.;
Browner, M. F. Insights into the conformational flexibility of Bruton’s
tyrosine kinase from multiple ligand complex structures. Protein Sci.
2011, 20, 428-436.
27. Bender, A. T.; Gardberg, A.; Pereira, A.; Johnson, T.; Wu, Y.;
Grenningloh, R.; Head, J.; Morandi, F.; Haselmayer, P.; Liu-Bujalski,
L. Ability of Bruton’s tyrosine kinase inhibitors to sequester Y551 and
prevent phosphorylation determines potency for inhibition of Fc
receptor but not B-cell receptor signaling. Mol. Pharmacol. 2017, 91,
208-219.
Z. K.; Soth, M.; McIntosh, J.; Litman, R.; Suh, J.; Kocer, B.; Davis, D.;
Park, J.; Frauchiger, S.; Dewdney, N.; Zecic, H.; Taygerly, J. P.; Sarma,
K.; Hong, J.; Hill, R. J.; Gabriel, T.; Goldstein, D. M.; Owens, T. D.
Structure-based drug design of RN486, a potent and selective Bruton’s
tyrosine kinase (BTK) inhibitor, for the treatment of rheumatoid
arthritis. J. Med. Chem. 2014, 58, 512-516.
12. Di Paolo, J. A.; Huang, T.; Balazs, M.; Barbosa, J.; Barck, K. H.;
Bravo, B. J.; Carano, R. A.; Darrow, J.; Davies, D. R.; DeForge, L. E.;
Diehl, L.; Ferrando, R.; Gallion, S. L.; Giannetti, A. M.; Gribling, P.;
Hurez, V.; Hymowitz, S. G.; Jones, R.; Kropf, J. E.; Lee, W. P.;
Maciejewski, P. M.; Mitchell, S. A.; Rong, H.; Staker, B. L.; Whitney,
J. A.; Yeh, S.; Young, W. B.; Yu, C.; Zhang, J.; Reif, K.; Currie, K. S.
Specific Btk inhibition suppresses B cell- and myeloid cell-mediated
arthritis. Nat. Chem. Biol. 2011, 7, 41-50.
13. Pan, Z.; Scheerens, H.; Li, S. J.; Schultz, B. E.; Sprengeler, P.
A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, K. C.;
Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg, L. A.;
Young, P. R.; Dalrymple, S. A.; Palmer, J. T. Discovery of selective
irreversible inhibitors for Bruton’s tyrosine kinase. ChemMedChem
2007, 2, 58-61.
14. Honigberg, L. A.; Smith, A. M.; Sirisawad, M.; Verner, E.;
Loury, D.; Chang, B.; Li, S.; Pan, Z.; Thamm, D. H.; Miller, R. A.;
Buggy, J. J. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-
cell activation and is efficacious in models of autoimmune disease and
B-cell malignancy. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 13075-
13080.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15. Byrd, J. C.; Furman, R. R.; Coutre, S. E.; Flinn, I. W.; Burger, J.
A.; Blum, K. A.; Grant, B.; Sharman, J. P.; Coleman, M.; Wierda, W.
G.; Jones, J. A.; Zhao, W.; Heerema, N. A.; Johnson, A. J.;
Sukbuntherng, J.; Chang, B. Y.; Clow, F.; Hedrick, E.; Buggy, J. J.;
James, D. F.; O’Brien, S. Targeting BTK with Ibrutinib in Relapsed
Chronic Lymphocytic Leukemia. N. Engl. J. Med. 2013, 369, 32-42.
16. Wang, M. L.; Simon, R.; Peter, M.; Andre, G.; Rebecca, A.;
Kahl, B. S.; Wojciech, J.; Advani, R. H.; Romaguera, J. E.; Williams,
M. E. Targeting BTK with Ibrutinib in Relapsed or Refractory Mantle-
Cell Lymphoma. N. Engl. J. Med. 2013, 369, 507-516.
28. Clinical arthritis scores from daily oral treatment from day 28 to
42 with different doses of compound 8 or Ibrutinib are presented as the
mean±SEM (n = 10). Disease control mice received vehicle only.
Scores from Ibrutinib (10 mg/kg) or 8 treatments at 3, 10 mg/kg were
significantly different compared with control (*p < 0.05, **p < 0.01,
***p<0.001 vs control).
17. Treon, S. P.; Tripsas, C. K.; Meid, K.; Warren, D.; Varma, G.;
Green, R.; Argyropoulos, K. V.; Yang, G.; Cao, Y.; Xu, L.; Patterson,
C. J.; Rodig, S.; Zehnder, J. L.; Aster, J. C.; Harris, N. L.; Kanan, S.;
Ghobrial, I.; Castillo, J. J.; Laubach, J. P.; Hunter, Z. R.; Salman, Z.;
Li, J.; Cheng, M.; Clow, F.; Graef, T.; Palomba, M. L.; Advani, R. H.
Ibrutinib in Previously Treated Waldenström’s Macroglobulinemia. N.
Engl. J. Med. 2015, 372, 1430-1440.
18. Caldwell, R. D.; Qiu, H.; Askew, B. C.; Bender, A. T.; Brugger,
T.; Camps, M.; Dhanabal, M.; Dutt, V.; Eichhorn, T.; Gardberg, A. S.;
Goutopoulos, A.; Grenningloh, R.; Head, J.; Healey, B.; Hodous, B. L.;
Huck, B. R.; Johnson, T. L.; Jones, C.; Jones, R. C.; Mochalkin, I.;
Morandi, F.; Nguyen, N.; Meyring, M.; Potnick, J. R.; Santos, D. C.;
Schmidt, R.; Sherer, B.; Shutes A.; Urbahns, K.; Follis, A. V.;
Wegener, A. A.; Zimmerli, S. C.; Bujalski, L. L. Discovery of
Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton’s
Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological
Diseases. J. Med. Chem. 2019, 62, 7643-7655.
19. Park, J. K.; Park, J. A.; Lee, Y. J.; Song, J.; Oh, J. I.; Lee, Y.-M.;
Suh, K. H.; Son, J.; Lee, E. B. THU0499ꢀHM71224, a novel oral BTK
inhibitor, inhibits human immune cell activation: new drug candidate
to treat B-cell associated autoimmune diseases. Ann. Rheum. Dis. 2014,
73, 355-356.
20. Yasuhiro, T.; Yoshizawa, T.; Birkett, J. T. P.; Kawabata, K.
ONO-4059, A novel Bruton’s tyrosine kinase (Btk) inhibitor:
synergistic activity in combination with chemotherapy in a ABC-
DLBCL Cell line. Blood 2013, 122, 5151-5151.
21. Evans, E. K.; Tester, R.; Aslanian, S.; Karp. R.; Sheets, M.;
Labenski, M. T.; Witowski, S. R.; Lounsbury, H.; Chaturvedi, P.;
Mazdiyasni, H.; Zhu, Z.; Nacht, M.; Freed, M. L.; Petter, R. C.;
Dubrovskiy, A.; Singh, J.; Westlin, W. F. Inhibition of Btk with CC-
292 provides early pharmacodynamic assessment of activity in mice
and humans. J Pharmacol Exp Ther. 2013, 346, 219-228.
22. Byrd, J. C.; Harrington, B.; O’Brien, S.; Jones, J. A.; Schuh, A.;
Devereux, S.; Chaves, J.; Wierda, W. G.; Awan, F. T.; Brown, J. R.;
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
O
4
5
6
NH2
N
N
HN
N
7
8
O
N
9
O
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IC50 (BTK) = 2.1 nM
The identification and lead optimization of a series of pyrazolo[3,4-d]pyridazinone derivatives are described as a novel class of
potent irreversible BTK inhibitors, resulting in the discovery of compound 8. Compound 8 exhibited high potency against BTK
kinase and acceptable PK profile. Furthermore, compound 8 demonstrated significant in vivo efficacy in a mouse collagen-induced
arthritis (CIA) model.
ACS Paragon Plus Environment