Design and Synthesis of Novel 3-(2-Aminopyridin-3-yl)-1,2,4-Triazolo[4,3-b]Pyridazine Derivatives as a Reversible Bruton's Tyrosine Kinase Inhibitors

Article abstract of DOI:10.1002/bkcs.11485

Bruton's tyrosine kinase, a non-receptor TEC family kinase, plays key role in B cell differentiation, proliferation, and survival. B cell receptor regulates the B cell's fate and cytokine release of B-lineage lymphoid leukemia cells which are deeply related with the pathogenesis of B-cell lineage of lymphoma and leukemia and autoimmune diseases. Thus, BTK protein regulation emerged as a promising therapeutic target for both various cancers and autoimmune diseases. Herein, we report the discovery of aminopyridin-1,2,4-triazolopyridazine derivatives as a reversible BTK inhibitor, and in vitro enzyme assay and cell based assay result were reported.

Full text of DOI:10.1002/bkcs.11485

Article
DOI: 10.1002/bkcs.11485
BULLETIN OF THE
C. H. Park et al.
KOREAN CHEMICAL SOCIETY
Design and Synthesis of Novel 3-(2-Aminopyridin-3-yl)-1,2,4-Triazolo
[4,3-b]Pyridazine Derivatives as a Reversible Bruton’s Tyrosine Kinase
Inhibitors
§
§
†
†
Chi Hoon Park,†,‡, Dukwoon Kim, Hyeonseok Jung, Jeong Hee Jeon, Raghavendra Achary,
*
Joo-Youn Lee,¶ Pilho Kim,†,‡ Heejung Jung,† Jong Yeon Hwang,†,‡ Do Hyun Ryu,§ Jae Du Ha,†
and Sung Yun Cho†,
*
†Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-606, South
Korea. *E-mail: sycho@krict.re.kr
‡Department of Medicinal and Pharmaceutical Chemistry, University of Science and Technology,
Daejeon 305-550, South Korea. *E-mail: chpark@krict.re.kr
§Department of Chemistry, Sungkyunkwan University, Suwon 440-746, South Korea
¶Korea Chemical Bank, Korea Research Institute of Chemical Technology, Daejeon 305-550, South
Korea
Received April 18, 2018, Accepted May 8, 2018
Bruton’s tyrosine kinase, a non-receptor TEC family kinase, plays key role in B cell differentiation, prolif-
eration, and survival. B cell receptor regulates the B cell’s fate and cytokine release of B-lineage lymphoid
leukemia cells which are deeply related with the pathogenesis of B-cell lineage of lymphoma and leuke-
mia and autoimmune diseases. Thus, BTK protein regulation emerged as a promising therapeutic target
for both various cancers and autoimmune diseases. Herein, we report the discovery of aminopyridin-
1,2,4-triazolopyridazine derivatives as a reversible BTK inhibitor, and in vitro enzyme assay and cell
based assay result were reported.
Keywords: Reversible Bruton’s kinase, BTK inhibitor, Triazolopyridazine derivative, Anticancer agent
Introduction
inhibitor approved in 2013 for the treatment of B-cell
malignancies, such as chronic lymphocyte leukemia and
mantle cell lymphoma. In clinical studies, ibrutinib
exhibited marked results in terms of the overall response
and survival rates of patients with several types of
mature B-cell malignancies. Other BTK inhibitors are
currently investigated in the early or late stages of clini-
cal trials (Figure 1). Among the BTK inhibitors tested in
these clinical trials, acalabrutinib is the most advanced
candidate at the late stages of clinical trials and approved
in 2017.
Another class of BTK inhibitors, BGB-3111 from Bei-
Gene (Beijing, China), is progressed to phase 3 clinical trial
in 2017. BTK inhibitors are now being attracted much
attention in autoimmune condition because BTK plays a key
role in B-cell’s fate. Acalabrutinib (AstraZeneca, Cambridge,
UK), BMS-986142 (Bristol-Meyers Squibb, New York,
USA), LY3337641 (Eli Lilly, Indianapolis, USA), Evobrutinib
(Merck KGaA, Darmstadt, Germany), RG7845 (Roche, Basel,
Swiss) are being developed in rheumatoid arthritis in phase
2 clinical trial. Evobrutinib and RG7845 are tested in phase
2 clinical trial for lupus treatment. Merck also tried in multiple
sclerosis, and Sanofi (Paris, France) is also engaged in phase
1 clinic trial for multiple sclerosis in 2017. Here, a novel series
of 3-(2-aminopyridin-3-yl-1,2,4-triazolo[4,3-b]pyridazine
derivatives substituted with various groups targeting BTK
Bruton’s tyrosine kinase (BTK) is a TEC family kinase,
which is a class of non-receptor kinases, and is expressed
in multiple cell types with tissue-specific expression in nor-
mal cells, including B lymphocytes, mast cells, and macro-
phages.1 BTK plays an important role in B-cell antigen
receptor (BCR) and Fc receptor signaling cascades with
low affinity, such as FcγRIII and FcγRIIa in monocytes.2
BTK has also been shown to play a role in models of auto-
immune diseases, such as collagen-induced arthritis and
lupus, which are closely related with BCR and Fcγ receptor
signaling pathways in aberrantly proliferated myeloid and
B-cells.3 The BCR signaling pathway regulates the activa-
tion, survival, proliferation, differentiation, and cytokine
release of B-lineage lymphoid leukemia cells.4 Dysregula-
tion of BTK is implicated in the pathogenesis of B-cell lin-
eage lymphoma and leukemia, due to the central roles of
BTK in the survival and proliferation of B-cells, which are
closely associated with B-cell malignancies and autoim-
mune diseases. Owing to the important roles of BTK in
hematological malignancies and autoimmune diseases,
BTK has emerged as an effective and attractive therapeutic
protein target.5 To date, a number of BTK inhibitors have
been developed and tested in the late stages of clinical tri-
als. Among the BTK inhibitors, ibrutinib is the only
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
1

Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
Scheme 2. Synthesis of 10 triazolopyridazine derivatives.
Reagents and conditions: (a) p-toluenesulfonic acid, toluene, 110
ꢀC, 18 h, 27%; (b) R1NH2, diisopropylethylamine, DMF, rt.,
48 h, 50%;
Figure 1. BTK inhibitors.
was investigated. In a previous study, we described the
discovery of a novel series of BTK inhibitors6.
Scheme 3. Synthesis of 10 triazolopyridazine derivatives.
Reagents and conditions: (a) 2,3-dimethoxybenzylamine, N-
methylpyrrolidone, 100 ꢀC, 18 h, 82%; (b) 4,4,5,5-tetramethyl-
Results and Discussion
2-aryl-1,3,2-dioxaborolane,
1
M
Na2CO3, Pd(PPh3)Cl2,
As a continuous work for identification of potent BTK
inhibitors, we initiated this study mainly focusing on the
development of reversible and selective BTK inhibitors
possessing triazolopyridazine scaffolds. A series of triazolo-
pyridazine derivatives were synthesized, and their biologi-
cal activities were evaluated. The general synthetic methods
adopted in the preparation of the triazolopyridazines ana-
logs are outlined in Scheme 1.
1,4-dioxane, 110 ꢀC, 18 h, 60%; (c) trifluoroacetic acid, 60 ꢀC,
18 h, 84%.
from 3,6-dichloropyridazine (3). Coupling of compounds
2 and 4 afforded the intermediate 5 in the presence of
triethylamine, as shown in Scheme 1. The intermediate
7 was readily obtained from 6 through the cyclization of
5 in the presence of p-toluenesulfonic acid under refluxing
condition in toluene at 110 ꢀC, followed by the suitable
alkyl amine substitution reaction with 6 as shown in
Scheme 2. The preparation method for the triazolopyrida-
zine derivative 10 is outlined in Scheme 3. Subsequent
treatment of 7 with 2,4-dimethoxybenzylamine at 100 ꢀC in
the presence of a base and palladium catalyzed the cross
In order to prepare 5-bromo-2-chloro-N0-(6-chloropyrida-
zin-3-yl)nicotinohydrazide (5), 5-bromo-2-chloronicotinic
acid (1) was converted into acyl chloride (2), and the requi-
site 3-chloro-6-hydrazinylpyridazine (4) was synthesized
Scheme 1. Synthesis of 5-bromo-2-chloro-N0-(6-chloropyridazin-
3-yl)nicotinohydrazide. Reagents and conditions: (a) SOCl2,
DMF, 80 ꢀC, 18 h, 52%; (b) NH2NH2, NH4OH, EtOH, 80 ꢀC,
4 h, 98%; (c) Et3N, CH2Cl2, rt., 18 h, 39%.
Scheme 4. Synthesis of 13 derivatives. Reagents and conditions:
(a) trifluoroacetic acid, 60 ꢀC, 18 h, 84%; (b) R3Cl or R3COCl,
NEt3, DMF, rt., 15 h, 65%.
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
2

Design and Synthesis of Novel 3-(2-Aminopyridin-3-Yl)-1,
BULLETIN OF THE
Article
2,4-Triazolo[4,3-b]Pyridazine Derivatives
KOREAN CHEMICAL SOCIETY
Table 1. Results of BTK inhibition and cell proliferation assays in the TMD8 cell line.7
Entry
R1
R2
BTK IC50 (μM)
TMD8 IC50 (μM)
10a
>10
>10
10b
10c
10d
1a
1a
>10
>10
>10
>10
>10
–NHMe (2a)
2.42
10e
10f
R3 = 1b
2a
2a
3.49
4.05
>10
>10
13a
2a
4.75
>10
10g
10h
13b
1a
1b
–NHCHMe2 (2b)
2b
2b
0.47
0.366
0.033
1.26
3.52
0.8
10i
2b
0.15
nd
10j
10k
10l
1e
1e
1e
1e
Aminocylohexane (2c)
Benzylamino (2d)
2c
Ph
0.174
0.71
0.34
0.17
nd
2.28
>10
10
10m
coupling of the resulting compound 8, readily affording the
intermediate 9. Subsequently, treatment of 9 with trifluoroa-
cetic acid afforded the triazolopyridazine derivative 10.
Modification of the piperidine group of 11 was performed
as a simple deprotection of the protective group and subse-
quent treatment with suitable electrophiles to produce the
alkylamines and amides (13 derivatives) as shown in
Scheme 4.
In an effort to further explore compounds that exhibit good
BTK inhibitory activity, the potency of aminopyridin-3-yl-1,-
2,4-triazolo[4,3-b]pyridazine derivatives, which were derived
from the focused library of triazolopyridazine scaffolds, was
examined in enzyme and cell cytotoxicity assays. All the com-
pounds were tested using an in vitro enzyme assay, whereas
cell cytotoxicity was evaluated in the cancer cell line TMD8.
In the cell-based assay, all the compounds were evaluated in a
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
3

Design and Synthesis of Novel 3-(2-Aminopyridin-3-Yl)-1,
BULLETIN OF THE
Article
2,4-Triazolo[4,3-b]Pyridazine Derivatives
KOREAN CHEMICAL SOCIETY
in pyrazole as shown in the synthesis of compound 10a. Intro-
duction of a hydroxyethyl group (10d) slightly improved the
enzyme activity relative to that of compound 5, without any
improvement observed in cell cytotoxicity. Next, groups bulk-
ier than hydroxyethyl-piperidine were introduced, such as in
10i–10m, as shown in Table 1. Among these compounds,
upon the introduction of isopropylamine (10i), the enzyme
activity increased similarly to that observed upon the introduc-
tion of a cyclohexylamine group (10j). However, substitution
with benzylamine (10k) was detrimental to the enzyme inhibi-
tory activity, whereas introduction of a polar substituent,
namely hydroxyethyl, was moderately effective in inhibiting
the enzyme activity, with no activity observed in the cell-
based assay. In addition, the enzyme inhibitory activity did
not significantly increase in the case of the phenylamino group
(10m). Furthermore, a methyl derivative of the parent com-
pound (13a) did not improve the inhibitory activity. However,
introduction of isopropylamine (13c, Table S1, Supporting
information) in place of methylamine (13a) improved both the
enzyme inhibitory activity and cell cytotoxicity. In the case of
cyclopentylamino, a bulkier group compound (13d, Table S1,
appendix S1) displayed an almost equivalent activity to that of
13c (Table S1, appendix S1). Replacement of the methyl
group in compound 13c (Table S1, appendix S1) with the
hydroxyethyl group (13b) markedly improved the enzyme
inhibitory activity and cell cytotoxicity as compared with
those reported for 10i.
Figure 2. Proposed binding model of the 13b (yellow, ball and
stick model) superimposed with X-ray binding conformation of
covalent inhibitor (orange, stick model) to BTK (blue ribbon
model).8 The BTK crystal structure in complexed with t-butyl cya-
noacrylamide inhibitor was obtained from protein data Bank
(PDB code 4YHF).9 The hydrogen bonds are displayed as a green
dashed lines. For clarity, only the key interaction residues sur-
rounding of the binding site are visible in stick model and are
labeled using the 3-letter amino acid code.
We performed docking study to understand the binding
mode of 13b compared with covalent bonding inhibitor
(Figure 2). As we expected, pyrazolo[3,4-d]pyrimidine scaf-
fold of 13b which is core scaffold to bind in the hinge region
makes hydrogen bonds with Met477 and Glu475. Also it is
located in hydrophobic pocket formed with Leu408, Ala428
and Leu528. The N-methyl-[1,2,4]triazolo[4,3-b]pyridazin-
6-amine forms hydrophobic interaction with Val416 and
Leu528. The pyrrole ring of 2-(4-(1H-pyrrol-1-yl)piperidin-
1-yl)ethan-1-ol group interact with Leu408 and the
2-(piperidin-1-yl)ethan-1-ol group extends toward a solvent
exposed area. Furthermore the terminal hydroxyl group can
make hydrogen bonding with backbone carbonyl of Ala478.
The use of aminocyclopentane (13e, Table S1, appendix S1)
in place of isopropylamine in compound 13b resulted in loss
of the enzyme inhibitory activity and cell cytotoxicity. To
further explore the importance of alkyl or alkanoyl groups to
piperidine, acetyl (13f, Table S1, appendix S1) and benzoyl
(13g, Table S1, appendix S1) groups were introduced. The
introduction of carbonyl groups to piperidine in compound
10i resulted in the loss of both enzyme activity and cell
cytotoxicity.
cell cytotoxicity assay conducted on the TMD8 cell line. The
TMD8 cell line is an activated B-cell (ABC)-type of diffuse
large B-cell lymphoma. TMD8 cells retain wild-type
CARD11 that plays major roles in aberrant BCR signaling in
the lymphomagenesis of B-cells. BTK shRNA exhibits high
toxicity against the TMD8 cell line and has been considered
as an appropriate cell line for ABC-type B-cell malignancy
screening. The results are summarized in Table 1. As the par-
ent compound 10e was identified as a relatively active com-
pound in the BTK enzyme assay, the synthesis of further
potent compounds was conducted through the structural modi-
fication of 10e. Introduction of an isopropyl (10g) instead of a
methyl (10e) group improved the BTK inhibitory activity,
with a marginal cytotoxic effect observed on the TMD8 cell
line. Moreover, with introduction of a relatively larger group
in comparison with the methyl or isopropyl groups, the cyclo-
hexylamine derivatives appeared to be detrimental, preventing
the increase in BTK enzyme inhibitory activities (10a). In
order to confirm whether the inhibitory activities of this scaf-
fold involved an irreversible or reversible mechanism, the
amino acrylate group was anchored to the third position of
cyclohexylamine. The results were not sufficient to determine
whether this scaffold was associated with a possibly irrevers-
ible mechanism, due to absence of inhibitory activity (10b,
10c). Instead, the present study focused on developing revers-
ible inhibitors without electrophile groups in the synthesized
compounds, which was achieved by the introduction of alkyl
In addition, we attempted to anchor acrylate to piperidine
in compound 13b to investigate whether this type of com-
pound could retain the inhibitory activity via an irreversible
mechanism or increase the inhibitory activity; however, the
results were unfavorable (13h, 13i, Table S1, appendix S1).
Other than the substituted pyrazole groups in this scaffold,
the study next focused on introducing aromatic rings
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
4

Design and Synthesis of Novel 3-(2-Aminopyridin-3-Yl)-1,
BULLETIN OF THE
Article
2,4-Triazolo[4,3-b]Pyridazine Derivatives
KOREAN CHEMICAL SOCIETY
4. A. J. Mohamed, L. Yu, C. M. Bäckesjö, L. Vargas, R. Faryal,
A. Aints, B. Christensson, A. Berglöf, M. Vihinen, B. F. Nore,
C. I. E. Smith, Immunol. Rev. 2009, 228, 58.
instead of pyrazoles. Substitution of a phenyl group with a
morpholinoethoxy group in the parent compound 6 did not
improve the activity. Other compounds such as 10n, 10o,
10p, 10q, and 10r in Table S1 (appendix S1) did not
exhibit any increase in their inhibitory activity, in terms of
both enzyme and cell-based activities. Introduction of a
substituent for the phenyl group resulted in loss of the
inhibitory activity (10s, Table S1, appendix S1).
5. (a) R. E. Davis, V. N. Ngo, G. Lenz, P. Tolar, R. M. Young,
P. B. Romesser, H. Kohlhammer, L. Lamy, H. Zhao, Y. Yang,
W. Xu, A. L. Shaffer, G. Wright, W. Xiao, J. Powell, J. K. Jiang,
C. J. Thomas, A. Rosenwald, G. Ott, H. K. Muller-Hermeling,
R. D. Gascoyne, J. M. Connors, N. A. Johnson, L. M. Rimsza,
E. Campo, E. S. Jaffe, W. H. Wilson, J. Delabie, E. B. Smeland,
R. I. Fisher, R. M. Braziel, R. R. Tubbs, J. R. Cook,
D. D. Weisenburger, W. C. Chan, S. K. Pierce, L. M. Staudt,
Nat. Lett. 2010, 463, 88. (b) A. Novero, P. M. Ravella, Y. Chen,
G. Dous, D. Liu, Exp. Hematol. Oncol. 2014, 3, 4.
Conclusions
In summary, novel aminopyridin-3-yl-1,2,4-triazolo[4,3-b]
pyridazine derivatives 13b and 13c showed potent in vitro
BTK enzyme inhibitory activity and TMD8 cell-based
assay. Further studies on the pharmacokinetics of 13b and
13c and those of their derivatives, as well as their effects in
in vivo animal models, will be conducted.
6. H. B. Park, C. H. Park, S.-T. Kang, J. H. Jeon, R. Achary, J.-
Y. Lee, P. Kim, H. Jung, C.-S. Yun, J. Y. Hwang, D. H. Ryu,
S. Y. Cho, Bull. Kor. Chem. Soc. 2017, 38, 278.
7. Bioassays. Enzyme and cell-based assay. Enzyme assay. The
experimental procedure was performed following the manufac-
turer’s instructions (Cisbio, Codolet, France). The reaction was
initiated by the addition of ATP to a mixture containing BTK,
peptide substrates, and inhibitors. After 30 min, an EDTA-
containing solution was added to stop the reaction. The EDTA
containing solution included Europium-conjugated antiphospho
residue antibody and streptavidine-XL665 (SA-XL665; Cisbio,
France) for the detection of the phosphorylated peptide
product. After 1 h of incubation, fluorescence was measured
using an Envision reader at 337 nm excitation and dual
665 and 620 nm emissions. The half maximal inhibitory
concentration (IC50) was calculated using GraphPad Prism
version 5 for Windows (La Jolla, CA, USA). The curves
were fitted using a nonlinear regression model with a log
(inhibitor) vs. response formula. Cell-based assay. For via-
bility experiments, TMD-8 cells were seeded in 96-well
plates at 30% confluency and exposed to chemicals. After
72 h, WST-1 reagent was added, and absorbance was mea-
sured at 450 nm using a Spectramax spectrophotometer
(Molecular Devices, Sunnyvale, CA, USA) according to the
manufacturer’s instructions. The IC50 values were calcu-
lated using GraphPad Prism version 5 for Windows. The
curves were fitted using a nonlinear regression model with a
log (inhibitor) vs. response formula.
Acknowledgments. This work was supported by a research
grant from National Research Council of Science and
Technology (NST) of the Korean government (CAP-15-
11-KRICT) and Korea Research Institute of Chemical
Technology.
Supporting Information. Additional supporting informa-
tion is available in the online version of this article
(Appendix SI).
References
1. (a) L. J. Crofford, L. E. Nyhoff, J. H. Sheehan, P. L. Kendall,
Expert. Rev. Clin. Immunol. 2016, 12, 763. (b) C. I. E. Smith,
B. Baskin, P. Humire-Greiff, J. N. Zhou, P. G. Olsson,
H. S. Maniar, P. Kjellen, J. D. Lambris, B. Christensson,
L. Hammarstrom, J. Immunol. 1994, 152, 557. (c) D. Vetrie,
I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter,
L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow,
C. I. E. Smith, D. R. Bentley, Nature 1993, 361, 226e233.
2. K. Pieper, B. Grimbacher, H. Eibel, J. Allergy Clin. Immunol.
2013, 131, 959.
8. Molecular modeling study of 13b against the BTK was per-
formed using the Schrödinger Suite 2017–4 (Schrödinger,
LLC, Cambridge, MA, USA; http://www.schrodinger.com).
The X-ray crystal structure obtained from the Protein Data
Bank (http://www.rcsb.org) that PDB code is 4YHF. The pro-
tein preparation was revised using Protein Preparation Wizard
in Maestro v.11.4 and the receptor grid box was generated
25 x 25 x 25 Å cubic size centered on complexed ligand. The
13b was minimized using an OPLS_2005 force field with a
dielectric constant value 80.0 in MacroModel v11.8. Flexible
ligand docking was performed using the Glide v.7.7 with stan-
dard precision method. The binding model of ligand was visu-
alized using Discovery Studio 2017 (Biovia, San Diego, CA,
USA; http://www.accelrys.com).
3. (a) J. A. Di Paolo, T. Huang, M. Balazs, J. Barbosa,
K. H. Barck, B. J. Bravo, R. A. Carano, J. Darrow,
D. R. Davies, L. E. DeForge, L. Diehl, R. Ferrando,
S. L. Gallion, A. M. Giannetti, P. Gribling, V. Hurez,
S. G. Hymowitz, R. Jones, J. E. Kropf, W. P. Lee,
P. M. Maciejewski, S. A. Mitchell, H. Rong, B. L. Staker,
J. A. Whitney, S. Yeh, W. B. Young, C. Yu, J. Zhang,
K. Reif, K. S. Currie, Nat. Chem. Biol. 2011, 7, 41.
(b) L. A. Honigberg, A. M. Smith, M. Sirisawad, E. Verner,
D. Loury, B. Chang, S. Li, Z. Pan, D. H. Thamm,
R. A. Miller, J. J. Buggy, Proc. Natl. Acad. Sci. USA 2010,
107, 13075. (c) M. S. Cohen, C. Zhang, K. M. Shokat,
J. Taunton, Science 2005, 308, 1318. (d) E. Leproult,
S. Barluenga, D. Moras, J. M. Wurtz, N. Winssinger, Med.
Chem. 2011, 54, 1347.
9. https://www.rcsb.org/structure/4YHF
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
5