Synthesis and evaluation of novel dimethylpyridazine derivatives as hedgehog signaling pathway inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.04.058

We report herein the design and synthesis of a series of structural modified dimethylpyridazine compounds as novel hedgehog signaling pathway inhibitors. The bicyclic phthalazine core and 4-methylamino-piperidine moiety of Taladegib were replaced with dim

Full text of DOI:10.1016/j.bmc.2018.04.058

Accepted Manuscript
Synthesis and evaluation of novel dimethylpyridazine derivatives as hedgehog
signaling pathway inhibitors
Chenglin Wang, Mingfei Zhu, Xiuhong Lu, Hong Wang, Weili Zhao, Xiongwen
Zhang, Xiaochun Dong
PII:
S0968-0896(18)30492-9
https://doi.org/10.1016/j.bmc.2018.04.058
BMC 14341
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 March 2018
23 April 2018
30 April 2018
Please cite this article as: Wang, C., Zhu, M., Lu, X., Wang, H., Zhao, W., Zhang, X., Dong, X., Synthesis and
evaluation of novel dimethylpyridazine derivatives as hedgehog signaling pathway inhibitors, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.058
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Synthesis and evaluation of novel dimethylpyridazine derivatives as
hedgehog signaling pathway inhibitors
Chenglin Wanga,§, Mingfei Zhub,§, Xiuhong Lua, Hong Wangb, Weili Zhaoa, Xiongwen
Zhangb,*, Xiaochun Donga,*
aDepartment of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai
201203, P. R. China
bShanghai Engineering Research Center of Molecular Therapeutics and New Drug
Development, School of Chemistry and Molecular Engineering, East China Normal
University, Shanghai 200062, P. R. China
KEYWORDS:
Hedgehog
signaling
pathway Inhibitors,
Anti-tumor
agents,
dimethylpyridazine,
ABSTRACT:
We report herein the design and synthesis of a series of structural modified
dimethylpyridazine compounds as novel hedgehog signaling pathway inhibitors. The bicyclic
phthalazine core and 4-methylamino-piperidine moiety of Taladegib were replaced with
dimethylpyridazine and different azacycle building blocks, respectively. The in vitro
Gli-luciferase assay results demonstrate that the new scaffold still retained potent inhibitory
potency. Piperidin-4-amine moiety was found to be the best linker between pharmacophores
dimethylpyridazine and fluorine substituted benzoyl group. Furthermore, the optimization of
1-methyl-1H-pyrazol and 4-fluoro-2-(trifluoromethyl)benzamide by different aliphatic or
aromatic rings were also investigated and the SAR were described. Several new derivatives
were found to show potent Hh signaling inhibitory activity with nanomolar IC50 values.

Among these compounds, compound 11c showed the highest inhibitory potency with an IC50
value of 2.33 nM, which was comparable to the lead compound Taladegib. In vivo efficacy of
11c in a ptch+/−p53−/− mouse medulloblastoma allograft model also indicated encouraging
results.
AUTHOR INFORMATION
Corresponding
authors.
E-mail:
xcdong@fudan.edu.cn
(X.
Dong),
xwzhang@sat.ecnu.edu.cn (X. Zhang).
§These authors contributed equally to this paper.
1. Introduction
The hedgehog (Hh) signaling pathway is an evolutionarily conserved signaling axis,
which is responsible for patterning and organogenesis in early embryonic development [1].
Under normal conditions, the secreted proteins Sonic hedgehog, Indian hedgehog and Desert
hedgehog bind to the negative regulator Patched (Ptch), relieving the suppression of Ptch to a
GPCR protein Smoothened (Smo). Smo activation triggers a series of intracellular events
ultimately lead to specific gene expression mediated by the Gli family transcription factors
[2]. However, mutational activation of Hh has been associated with a variety of cancers, such
as basal cell carcinoma (BCC), medulloblastoma (MB), lung, colorectal, prostate, pancreatic,
breast and some blood cancers [1,3]. Furthermore, the fact that pharmacological inhibition of
Hh signaling could impair the growth of imatinib-resistant mouse and human chronic
myelogenous leukemia (CML) makes this signaling pathway attractive to researchers because
of the frequent drug-resistant in clinical tumor therapy [4]. Therefore, inhibition of the
aberrant Hh signaling represents a promising approach for novel anticancer therapy [3].

Since the natural steroidal alkaloid cyclopamine was identified as the first Smo
antagonist to block Hh signaling by directly binding to Smo [5], a number of Smo-targeting
small molecules have been developed in recent years. Two agents, Vismodegib (GDC-0449,
1) and Sonidegib (LDE-225, 2), have been approved by FDA in 2012 and 2015 for treatment
of locally advanced basal cell carcinoma (BCC) [6,7]. Inꢀaddition, several Hh inhibitors are
now in different stages of development including LEQ-506 (phase I, 3) [8], LY-2940680
(Taladegib, phase II, 4) [9], IPI-926 (Saridegib, phase II), XL-139 (phase II), PF-04449913
(Glasdegib, phase II) [10] and Itraconazole (phase II) [11].
Figure 1. Chemical structure of several Hh signaling pathway inhibitors
During our investigation of novel promising Hh antagonists, we envisioned that the
structural modification of Taladegib, which was in an active stage of clinical development,
might lead to a new scaffold with better potency and physiochemical properties [9]. The
co-crystal structures of the transmembrane domain of the human Smo receptor bound to the
Taladegib was reported by Stevens’s group in 2013 [12], which have provided a rich set of
structural information for drug discovery efforts on Hedgehog pathway inhibitors. Based on
the solved co-crystal structure, Taladegib forms three critical hydrogen bonds binding with
Smo protein: one hydrogen bond from the amide oxygen atom to N219 residue, two
hydrogen bonds between the phthalazine core and R400. Moreover, the

4-fluoro-2-trifluoromethylphenyl moiety of Taladegib forms extensive interactions with
residues from ECL3, including Q477, W480, E481 and F484 which stacks to the phenyl ring
of Taladegib through π−π interaction. The 4-methylaminopiperidine ring of Taladegib plays
an important role as a linker to adjust the orientation of pharmacophores phthalazine and
fluorine substituted benzoyl group. [13]
Previously, we have reported our medicinal chemistry efforts on the discovery of novel
highly potent Hh inhibitors, which contained a central backbone of phthalazine [14,15]. The
piperazine linker of lead compound Anta XV (5) was replaced by different four, five and six
heterocyclic or spirocyclic building blocks. The most potent compound with
piperidin-4-amine moiety was found to possess subnanomolar activity for antagonizing Hh
pathway, which was about 12-fold of the potency of Anta XV. This compound also possesses
potent antitumor activities both in cell-based assay and in vivo allograft studies [14].
Furthermore, we had applied the similar strategy to replace the 4-methylamino-piperidine
moiety of Taladegib with different four, five or six-membered azacycle or azaspirocycle
building blocks. Several derivatives were found to display potent antitumor activities both in
in vitro and in vivo studies [15].
LEQ-506 (3) is another potent second–generation Smo inhibitor discovered by Novartis.
The bicyclic phthalazine core of Anta XV (5) was replaced with dimethylpyridazine to yield
the compound with better potency, decreased hERG activity and improved aqueous solubility
[16]. In this study, we choose Taladegib as our lead compound for the development of novel
Hh signaling pathway inhibitors. Based on the strategy that Novartis adopted and our early
experience in Taladegib modification, we tried to take advantage of the dimethylpyridazine
skeleton as a privileged scaffold for phthalazine surrogate and replaced the
4-methylamino-piperidine moiety with different five or six-membered azacycle building
blocks to adjust the orientation of pharmacophores dimethylpyridazine and fluorine

substituted benzoyl group (Figure 2, region B). A series of structural modified novel
dimethylpyridazine compounds were prepared. Furthermore, the optimization of region A
and region C by different aliphatic or aromatic rings were also investigated. Herein, the
syntheses and preliminary evaluations in vitro are reported.
Figure 2：Design concept of novel dimethylpyridazine derivatives.
2. Chemistry
The first round of modification focused on the central linking region B to replace
4-methylamino-piperidine
moiety
with
piperazine,
3-amino-pyrrolidine
or
4-amino-piperidine. Four structural modified novel dimethylpyridazine compounds 11a−c
and 12 were prepared. As shown in Scheme 1, 3,6-dichloro-4,5-dimethylpyridazine (7) was
reacted with N-Boc protected five and six-membered azacyclic building blocks 6a−c to give
compounds 8a−c. After deprotection with trifluoroacetic acid, the obtained amine 9a−c were
then condensed with 4-fluoro-2-(trifluoromethyl)benzoic acid under conventional
condensation conditions (HATU, DIPEA, DCM) to give compounds 10a−c. The target
molecules 11a−c were finally synthesized through Suzuki coupling using 10a−c reacted with
1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole in the presence of
Pd(PPh3)4 in m-xylene assisted by microwave irradiation. Compound 11c were then
methylated with iodomethane to afford the compound 12.

Scheme 1. Synthesis of the compounds 11a-c and 12: Reagents and conditions: (a) K2CO3,
DMF, 110˚C, 7 h, 48–84%; (b) TFA, DCM, rt, 2 h; (c) 4-fluoro-2-(trifluoromethyl)benzoic
acid,
HATU,
DIPEA,
DCM,
rt,
6
h,
53–76%;
(d)
1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(PPh3)4, K3PO4,
KF, m-xylene, microwave 120 °C, 1 h, 31–35%; (e) CH3I, NaH, THF, rt, 2 h, 36%.
To our delight, quick assay evaluation (see Results and discussion) revealed that
dimethylpyridazine derivative with piperidin-4-amine linker (11c) led to comparable activity
to Taladegib. Thus, the second round of modification fixed the linker region B as
piperidin-4-amine and focused on the change in region A. Various aromatic rings were used
to replace the 1-methyl-1H-pyrazol moiety. The synthesis of designed compounds 13a−g was
described in Scheme 2. The above chloride 10c reacted with different aryl boric acid esters
under Suzuki coupling conditions assisted by microwave irradiation in the presence of
Pd(PPh3)4, K3PO4 and KF to afford target molecules 13a−g.
Scheme 2. Synthesis of the compounds 13a-g: Reagents and conditions: (a) ArB(OH)2,
Pd(PPh3)4, K3PO4, KF, m-xylene, microwave 120 °C, 1 h, 30–39%.

Unfortunately, the replacement of 1-methyl-1H-pyrazole by different aromatic ring
afforded reduced inhibitory effect. Therefore, we retained 1-methyl-1H-pyrazole moiety and
focused on the change in region C. As depicted in Scheme 3, the N-Boc protected chloride 8c
reacted with 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole under
Suzuki coupling conditions assisted by microwave irradiation to afford compound 14. After
deprotection, intermediate 15 was then reacted with chlorinated aryl esters in NMP at 150 °C
with microwave irradiation to afford corresponding esters. Treatment of these esters with
CH3MgI led to the target molecules 16a–c. And target molecule 16d was obtained by
N-arylation of intermediate 15 with 2-bromo-5-(methylsulfonyl)pyrazine in NMP.
Scheme 3. Synthesis of the compounds 16a–d: Reagents and conditions: (a)
1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(PPh3)4, K3PO4,
KF, m-xylene, microwave 120 °C, 1 h, 53%; (b) TFA, DCM, rt, 2 h; (c) methyl
6-chloronicotinate for 16a (methyl 5-chloropyrazine-2-carboxylate for 16b, methyl
6-chloropyridazine-3-carboxylate for 16c), Et3N, NMP, microwave 150 °C, 0.5 h; (d)
CH3MgI, THF, rt, 6 h, 10–22%; (e) 2-bromo-5-(methylsulfonyl) pyrazine, Et3N, NMP,
microwave 150 °C, 0.5 h, 18%.
Meanwhile, we tried to introduce urea, carbamate or amide linkage to connect region B
and region C. The synthesis of designed compounds 18a–d, 19, 20 and 21a–e was described
in Scheme 4. Compound 15 was reacted with 17a–d prepared via one step described in
Scheme 5 to afford compounds 18a–d. Target molecules 19 was obtained by condensation of
15 with phenyl carbonochloridate in the presence of pyridine. Condensation of 15 with
cyclohexyl (4-nitrophenyl) carbonate generated the target compound 20. Finally, target

compounds 21a–e were prepared by condensation with corresponding benzoic acid under
aforementioned standard conditions.
Scheme 4. Synthesis of the compounds 18a–d, 19, 20 and 21a–e: Reagents and conditions: (a)
17a-d, Et3N, DMSO, 100 °C, 8 h, 20-28%; (b) phenyl carbonochloridate, pyridine, DCM, rt,
6 h, 20%; (c) cyclohexyl (4-nitrophenyl) carbonate, DIPEA, THF, rt, 6 h, 16%; (h)
corresponding benzoic acid, HATU, DIPEA, DCM, rt, 6 h, 27–43%.
Scheme 5. Synthesis of the compounds 17a–d: Reagents and conditions: (a) corresponding
amine, pyridine, DCM, rt, 5 h, 65-80%.
Recently, Xu and collaborators have reported the crystal structure of the multi-domain
human Smo, which may inspire the design of a new type of small molecule that links the
cysteine-rich domain (CRD) and seven-transmembrane helices domain (TMD), or interacts
with the hinge domain (HD) to regulate domain-domain communications that can potentially
exert amplified efficacy and overcome drug resistance [17]. Therefore, our next effort was to
verify if phenyl-substituted compounds could exhibit moderately enhanced stabilization on
Smo [18]. The synthesis of designed compounds 23a–c was described in Scheme 6. These
compounds were prepared in 33−40% yields by treating compound 11c with phenol or

thiophenol under strong basic condition.
Scheme 6. Synthesis of the compounds 24a-c: Reagents and conditions: (a) phenol or
thiophenol, t-BuOK, DMSO, 120 °C, 6 h, 33–40%.
3. Results and discussion
3.1. Hh signaling inhibitory activities of the synthesized compounds and the structure-activity
relationships (SAR).
All new compounds were evaluated for their ability to inhibit the Hh signaling pathway
by dual luciferase reporter assays using light II cells, which were NIH-3T3 cells stably
transfected with a Gli-responsive firefly luciferase reporter and Renilla-luciferase expression
vector [14,15]. The inhibition rate at 100 nM drug concentration were evaluated first and IC50
values were then determined if the inhibition was more than 50%.
The in vitro IC50 values of compounds 11a–c and 12 were illustrated in Table 1. Both
Vismodegib (GDC–0449, 1) and Taladegib (4) were used as positive controls. As expected,
replacement of 4-methylamino-piperidine with different five or six-membered heterocyclic
building blocks led to significant influences on Hh pathway inhibition. Three of the
synthesized compounds exhibit Hedgehog signaling pathway inhibition with IC50 values less
than 100 nM. To our delight, a change from phthalazine to dimethylpyridazine was
well-tolerated (11c vs 4), and the similar inhibition activity was observed (IC50 = 2.33 nM).
However, N-methylation of compound 11c resulted in decreased potency (12 vs 11c), with
IC50 value of 29.99 nM. A diminished activity was observed upon replacement of the
4-methylamino-piperidine by pyrrolidin-3-amine (11a), with IC50 value of 36.39 nM. The

change from 4-methylamino-piperidine to piperazine resulted in the totally loss of activity
(11b).
Table 1. Inhibition of Compounds to the Hh Pathway Activity Tested by Dual Luciferase
Reporter Assays in Light II Cells
Compounds
core
Inhibition at 100 nM(%)
54.13
Gli-luc reporter IC50 (nM) a
36.39 ± 5.53
11a
-6.57
>100
11b
89.49
60.87
2.33 ± 0.22
11c
12
29.99 ± 4.23
Taladegib (4)
94.99
2.26 ± 0.36
2.46 ± 0.50
Vismodegib (1)
108.02
a Values represent mean ± standard error of three measurements.
Since compound 11c with dimethylpyridazine moiety was found to possess nanomolar
activity in Gli-luciferase reporter assay, which is almost the same as the lead compound
Taladegib (4). Further optimization of 1-methyl-1H-pyrazole component within the
dimethylpyridazine derivative 11c was conducted and the compounds 13a–g were evaluated
for their ability to inhibit the Hh signaling pathway. As shown in Table 2, two of the
synthesized compounds exhibit inhibitory potency with IC50 values less than 10 nM.
Replacement of 1-methyl-1H-pyrazole by a phenyl ring afforded reduced inhibitory effect
(11c vs 13a). Both electron-withdrawing group and electron-releasing group substitutions
showed reduced inhibitory effect (compounds 13b–f), compared to compound 11c. Only the

p-cyano or o-chloro substitution show just slightly decreased activity (11c vs 13c and 13f),
with IC50 value of 7.02 nM and 8.50 nM, respectively. Meanwhile, replacement of
1-methyl-1H-pyrazole by pyridine also diminished the potency, with about 20-fold drop in
activity (11c vs 13g).
Table 2. Inhibition of Compounds to the Hh Pathway Activity Tested by Dual Luciferase
Reporter Assays in Light II Cells
Inhibition at
100 nM(%)
Gli-luc reporter
IC50 (nM) a
Compounds
R1
R2
13a
90.17
25.52 ± 4.67
13b
84.99
43.78 ± 1.07
7.02 ± 0.58
106.32
13c
13d
13e
101.41
12.15 ± 7.54
74.52
21.96 ± 2.13
8.50 ± 1.67
110.94
13f
75.97
31.84
10.82
43.18 ± 8.66
>100
13g
16a
16b
>100

20.61
>100
16c
16d
18a
23.80
24.35
>100
>100
18b
18c
18d
19
45.73
26.23
3.47
>100
>100
>100
41.19
26.65
81.96
>100
20
>100
21a
61.54 ± 7.64
21b
21c
21d
79.80
79.66
-14.62
42.91 ± 4.46
32.84 ± 2.38
>100
21e
82.11
25.10 ± 3.30
a Values represent mean ± standard error of three measurements.
The replacement of 1-methyl-1H-pyrazole with different aromatic ring afforded reduced
inhibitory effect, thus we retained 1-methyl-1H-pyrazole moiety and focused on the change in

region C. Considering that pyridin-3-propanol moiety of ANTA XV (5) can form two
hydrogen bonds with Smo protein [13], we changed the 4-fluoro-2-trifluoromethylbenzoyl
group of compound 13c with pyridin-3-propanol, pyrazin-2-propanol, pyridazin-3-propanol
and 2-methylsulfonylpyrazine to generate four hybrid compounds. Unfortunately, the four
new compounds 16a–d showed rather weak inhibitory effects at the concentration of 100 nM
Gli-luciferase reporter assay (Table 2). Meanwhile, we used urea and carbamate to connect
region B and region C instead of amide. As shown in Table 2, replacement of the amide
moiety with urea and carbamate afforded compound 18a–d, 19 and 20 exhibited negligible
activity as well. These results may indicate that the huge modification of region C is not
tolerated. Therefore, further optimization of region C component within the amide was
conducted and the compounds 21a–e were evaluated for their ability to inhibit the Hh
signaling pathway. The 5-fluoro-2-(trifluoromethyl)benzamide substituted analog 21a and
6-fluoro-2-(trifluoromethyl)benzamide substituted analog 21b showed reduced inhibition
against the Hh signaling pathway with an IC50 value of 61.54 nM and 42.91 nM, respectively.
The replacement of the 4-fluoro-substituent with chlorine resulted in a 10-fold drop in
activity (11c vs 21e). The presence of a trifluoromethyl substituent on the aromatic ring,
proved to be essential to activity. This strong electron-withdrawing group likely enhanced the
π–π interaction with Phe484. Removal of the trifluoromethyl from the 2-position (21d)
resulted in totally loss of activity, whereas removal of the fluoro from the 4-position (21c) led
to just 15-fold decrease in inhibition of the Hh pathway.
Introduction of a phenyl ether into Taladegib can strengthen its binding and stabilize the
Smo [18], which may inspire the design of new inhibitor that potentially exert amplified
efficacy and overcome drug resistance. To further determine the effect of the
phenyl-substituted compounds on the Hh pathway activity, compounds 23a−c were designed
and tested (Table 3). Gratifyingly, this small series of compounds retained part potency

against the Hh signaling pathway, compounds 23a and 23b showing IC50 values of 36.47nM
and 16.61 nM, respectively. Another thiophenol substituted analogue (23c) also retained the
potency of 32.46 nM.
Table 3. Inhibition of Compounds to the Hh Pathway Activity Tested by Dual Luciferase
Reporter Assays in Light II Cells
Compounds
X
O
R
H
Gli-luc reporter 100 nM(%) Gli-luc reporter IC50 (nM) a
78.90
36.47 ± 5.76
23a
O
S
F
91.48
82.82
16.61 ± 0.99
32.46 ± 1.89
23b
23c
H
a Values represent mean ± standard error of three measurements.
3.2. In vivo efficacy evaluation of compound 11c
Encouraged by the potent inhibitory activity of compound 11c on the Gli-luciferase
reporter assay, we were eager to know whether the excellent inhibition can be translated into
favorable anti-tumor effect. To this end, we focused on the Ptch+/–p53–/– medulloblastoma
mouse model, a well-accepted Hh-driven mouse medulloblastoma model that is routinely
used to test the efficacy of Hh inhibitors [15]. When the volume of tumors reached around
150–200 mm3, the tested compound was administered at 10 mg/kg or 20 mg/kg by oral
gavage twice a day. As shown in Figure 3, compared to the vehicle control, compound 11c
obviously inhibited the growth of allografted medulloblastoma. And the in vivo efficacy of
11c is slightly more active than the marketed drug Vismodegib (1). In addition, 11c was well
tolerated, affording no significant body weight loss at the dose investigated (Figure S1,
Supporting Information).

Figure 3. Antitumor activity upon treatment with 11c, Vismodegib (1) or vehicle in
Ptch+/–p53–/– medulloblastoma allograft model. Vehicle: 0.5% NaCMC suspension. Tumor
volume for indicated days was showed as means ± SEM (n = 5).
3.3. Molecular Docking and Simulation
Molecular docking study was performed to elucidate the binding model of most active
compound 11c into the three-dimensional Smo complex structure (4JKV.pdb) using
Schrodinger (Maestro suite). As shown in Figure 4, 11c forms three critical hydrogen bonds
with Smo protein: one hydrogen bond from the amide oxygen atom to Asn219 residue, two
hydrogen bonds between the dimethylpyridazine core and Arg400. Furthermore, phenyl ring,
pyridazine ring and pyrazol ring interacted with the electron-rich benzene ring of Phe484,
Tyr394 and Trp281 via π-π stacking respectively. The docking results demonstrated that the
key interactions derived from the Taladegib−Smo crystal complex were almost reserved by
our scaffold optimizations. More importantly, 11c (orange carbons) bind in almost the same
location as that of Taladegib (yellow carbons). All these computational predictions suggested
that 11c can exactly interact with ligand binding cavity of Smo and the in silico experiments
corroborate well with in vitro results.

Figure 4. Key interactions of 11c in the active site of Smo and superposition of 11c (orange)
and Taladegib (yellow). The pictures were generated using Pymol.
4. Conclusion
In summary, a series of structural modified dimethylpyridazine compounds were
designed and synthesized. The in vitro Gli-luciferase assay results demonstrate that the new
scaffold still retained potent inhibitory potency. Piperidin-4-amine moiety was found to be
the
best
linker
between
pharmacophores
dimethylpyridazine
and
4-fluoro-2-(trifluoromethyl)benzamide. The expanded comprehensive SAR on region A and
C were also investigated and several derivatives were found to show potent Hh signaling
inhibitory activity. Among all these compounds, compound 11c showed the highest
inhibitory potency with an IC50 value of 2.33 nM, which was comparable to the lead
compound Taladegib. Furthermore in vivo allograft studies of 11c also indicated encouraging
results.
5. Experimental
5.1. Chemistry

General. All chemicals were purchased from Adamas, SCRC, Alfa Aesar, Acros and used
without further purification. Deuterated solvents were purchased from Cambridge Isotope
Laboratories. All non-aqueous reactions were carried out using oven-dried (110 °C) or heat
gun dried glassware under a positive pressure of dry argon unless otherwise noted. THF and
dichloromethane were purified by distillation and dried by passage over activated molecular
1
sieves (type 4 Å) under an argon atmosphere. H and 13C NMR data were recorded on a
Varian Model Mercury 400 MHz and Bruker 600 MHz spectrometers using solvent signals
1
(DMSO-d6: δH 2.50/δC 39.5; CDCl3: δH 7.26/δC 77.2) as references. H NMR chemical shifts
(δ) are given in ppm (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) downfield
from Me4Si. LC-MS data were recorded on an Agilent 1260/6120 quadrupole LC/MS
spectrometer, and High resolution mass spectra obtained on an AB SCIEX Triple TOFTM
5600+ mass spectrometer.
5.1.1. General procedure for the synthesis of compounds 8a–c
Solid K2CO3 (2 equiv) was added to a solution of 3,6-dichloro-4,5-dimethylpyridazine
(1 equiv) and amine (1 equiv) in DMF (20 mL), and the resulting solution was stirred at
110 °C for 7 h. The reaction mixture was concentrated under reduced pressure and dissolved
in EtOAc, washed with water, dried over magnesium sulfate, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (ethyl acetate: hexane
= 4:1) to yield pure product.
5.1.1.1. tert-butyl (R)-(1-(6-chloro-4,5-dimethylpyridazin-3-yl)pyrrolidin-3-yl)carbamate (8a).
Yellow solid (84%); 1H NMR (400 MHz, CDCl3) δ 4.89 (s, 1H), 4.29 (s, 1H), 3.80–3.61 (m,
2H), 3.57–3.36 (m, 2H), 2.30 (s, 3H), 2.24 (s, 3H), 1.96–1.84 (m, 1H), 1.69 (s, 1H), 1.43 (s,
9H); MS (ESI+): [M+H]+: 327.2.

5.1.1.2. tert-butyl 4-(6-chloro-4,5-dimethylpyridazin-3-yl)piperazine-1-carboxylate (8b).
White solid (48%); 1H NMR (400 MHz, CDCl3) δ 3.57 (dd, J = 6.1, 4.0 Hz, 4H), 3.15 (s, 4H),
2.32 (s, 3H), 2.26 (s, 3H), 1.47 (s, 9H); MS (ESI+): [M+H]+: 327.2.
5.1.1.3. tert-butyl (1-(6-chloro-4,5-dimethylpyridazin-3-yl)piperidin-4-yl)carbamate (8c).
Light yellow solid (80%); 1H NMR (400 MHz, CDCl3) δ 4.55 (s, 1H), 3.69 (s, 1H), 3.43 (d, J
= 12.9 Hz, 2H), 3.03 (t, J = 11.7 Hz, 2H), 2.33 (s, 3H), 2.25 (s, 3H), 2.08 (d, J = 10.8 Hz, 2H),
1.59 (d, J = 10.8 Hz, 2H), 1.47 (s, 9H); MS (ESI+): [M+H]+: 341.2.
5.1.2. General procedure for the synthesis of compounds 9a–c
Amide (1 equiv) was dissolved in dichloromethane (20 mL) and charged with TFA (6
equiv) in drops. The mixture was stirred at room temperature for 5 h, and was concentrated
afterwards. Without further purification, the crude material was used directly into the next
reaction.
5.1.3. General procedure for the synthesis of compounds 10a–c
DIPEA (0.5 mL) was added to a solution of amine (1 equiv) in dichloromethane (10
mL). The mixture was stirred at 0˚C for 15min. Then 4-fluoro-2-(trifluoromethyl)benzoic
acid (1.2 equiv) and HATU (1.2 equiv) were added to the solution, the reaction mixture was
stirred for 6 h while warming at room temperature. The reaction solution was washed with
water and extracted with dichloromethane, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel column chromatography
(dichloromethane: methanol = 30:1) to yield pure product.
5.1.3.1.
(R)-N-(1-(6-chloro-4,5-dimethylpyridazin-3-yl)pyrrolidin-3-yl)-4-fluoro-2-(trifluoromethyl)b
enzamide (10a). Yellow solid (76%); 1H NMR (400 MHz, CDCl3) δ 7.51 (dd, J = 8.5, 5.4 Hz,
1H), 7.41 (d, J = 7.0 Hz, 1H), 7.35 (dd, J = 8.8, 2.5 Hz, 1H), 7.16–7.11 (m, 1H), 4.71 (s, 1H),

3.80 (dd, J = 11.2, 5.3 Hz, 1H), 3.70 (d, J = 11.0 Hz, 1H), 3.65–3.56 (m, 1H), 2.98–2.93 (m,
1H), 2.24 (s, 3H), 2.20 (s, 3H), 2.15 (dd, J = 8.0, 4.8 Hz, 2H); MS (ESI+): [M+H]+: 417.2.
5.1.3.2. (4-(6-chloro-4,5-dimethylpyridazin-3-yl)piperazin-1-yl)(4-fluoro-2-(trifluoromethyl)
phenyl)methanone (10b). White solid (53%); 1H NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 8.7,
2.4 Hz, 1H), 7.38 (dd, J = 8.5, 5.4 Hz, 1H), 7.35–7.30 (m, 1H), 4.10–3.97 (m, 1H), 3.91–3.85
(m, 1H), 3.39–3.35 (m, 2H), 3.27–3.17 (m, 4H), 2.34 (s, 3H), 2.28 (s, 3H); MS (ESI+):
[M+H]+: 417.5.
5.1.3.3. N-(1-(6-chloro-4,5-dimethylpyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluoromethyl)
benzamide (10c). Yellow solid (yield 66%); 1H NMR (400 MHz, CDCl3) δ 7.58 (dd, J = 8.4,
5.5 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.30 (t, J = 8.2 Hz, 1H), 5.84 (d, J = 8.0 Hz, 1H), 4.22
(d, J = 10.5 Hz, 1H), 3.48 (d, J = 13.1 Hz, 2H), 3.11 (t, J = 11.9 Hz, 2H), 2.33 (d, J = 1.5 Hz,
3H), 2.26 (d, J = 2.5 Hz, 3H), 2.18 (d, J = 13.0 Hz, 2H), 1.74–1.68 (m, 2H); MS (ESI+):
[M+H]+: 431.2.
5.1.4. General procedure for the synthesis of compounds 11a–c
Amide pyridazine (1 equiv), borate ester (2 equiv), potassium fluoride (2 equiv),
potassium phosphate (2 equiv) and m-xylene (5 mL) were added to a microwave vial (20 mL)
equipped with a stir bar. The reaction mixture was purged with nitrogen for 2 min.
Tetrakis(triphenylphosphine)platinum (0.1 equiv) was added and purged with nitrogen for 2
min again. The vial was sealed and irradiated in the microwave at 120 °C (high absorption
setting, Biotage Initiator) for 3 h. The reaction mixture was concentrated under reduced
pressure and dissolved in EtOAc (20 mL), washed with water, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The residue was purified by silica gel column
chromatography (dichloromethane: methanol = 50:1) to yield pure product.
5.1.4.1.
(R)-N-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)pyrrolidin-3-yl)-4-fluoro-

1
2-(trifluoromethyl)benzamide (11a). Yellow solid (31%); H NMR (400 MHz, CDCl3) δ
7.58–7.47 (m, 2H), 7.34 (dd, J = 8.8, 2.5 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 6.61 (d, J = 7.0
Hz, 1H), 6.28 (d, J = 1.9 Hz, 1H), 4.78–4.66 (m, 1H), 3.87 (dd, J = 11.4, 5.6 Hz, 1H), 3.80 (s,
3H), 3.76–3.71 (m, 1H), 3.61–3.53 (m, 1H), 2.25 (s, 3H), 2.16 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 166.70, 160.67, 147.63, 138.47, 138.06, 137.17, 131.95, 131.21 (d, J = 8.2), 126.10,
119.05 (d, J = 21.2), 114.13, 113.99, 107.68, 55.27, 50.47, 48.59, 37.60, 31.59, 15.92, 15.32.
HRMS (ESI) calcd for C22H22F4N6O [M+H]+ 463.1864, found 463.1887.
5.1.4.2.
(4-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperazin-1-yl)(4-fluoro-2-(trifl
1
uoromethyl)phenyl)methanone (11b). Yellow solid (33%); H NMR (400 MHz, CDCl3) δ
7.57 (d, J = 2.0 Hz, 1H), 7.47–7.30 (m, 3H), 6.37 (d, J = 1.9 Hz, 1H), 4.10–4.02 (m, 1H),
3.96 (dd, J = 7.3, 3.5 Hz, 1H), 3.92 (s, 3H), 3.39–3.43 (m, 2H), 3.37–3.27 (m, 4H), 2.31 (s,
13
3H), 2.24 (s, 3H); C NMR (151 MHz, CDCl3) δ 166.71, 163.10, 162.14, 161.44, 149.93,
137.97, 130.77, 129.91, 129.70, 129.52 (d, J = 8.2), 119.48, 114.49, 108.02, 50.22, 49.44,
47.12, 41.72, 37.90, 16.12, 14.48; HRMS (ESI) calcd for C22H22F4N6O [M+H]+ 463.1864,
found 463.1867.
5.1.4.3.
N-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(tri
1
fluoromethyl)benzamide (11c). White solid (35%); H NMR (400 MHz, CDCl3) δ 7.62–7.55
(m, 2H), 7.41 (d, J = 8.7 Hz, 1H), 7.31–7.26 (m, 1H), 6.36 (d, J = 1.8 Hz, 1H), 5.81 (d, J =
7.7 Hz, 1H), 4.28–4.21 (m, 1H), 3.92 (s, 3H), 3.60 (d, J = 13.3 Hz, 2H), 3.22–3.15 (m, 2H),
13
2.29 (s, 3H), 2.22 (s, 3H), 2.20 (s, 2H), 1.80–1.70 (m, 2H); C NMR (151 MHz, CDCl3) δ
165.55, 162.17, 148.65, 137.60, 137.36, 136.80, 131.60, 130.54 (d, J = 8.3), 129.09, 118.51,
118.37, 113.51, 113.34, 107.26, 48.44, 46.72, 37.22, 31.07, 15.41, 13.97; HRMS (ESI) calcd
for C23H24F4N6O [M+H]+ 477.2020, found 477.2040.

5.1.5.
N-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)-4-fluoro-N-m
ethyl-2-(trifluoromethyl)benzamide (12)
N-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)-4-fluoro
-2-(trifluoromethyl)benzamide (11c) (100 mg, 0.21 mmol), and NaH (10 mg, 0.42 mmol)
were added to THF (20 mL) under nitrogen. The obtained solution was cooled down on an
ice-water bath. CH3I (60 mg, 0.42 mmol) was added to the solution, and the reaction mixture
was stirred for 2 h while warming at room temperature. Then the reaction was quenched by
addition of saturated NH4Cl, and the organics were extracted with EtOAc. The organic layers
were dried over MgSO4, filtered, and concentrated. The residue was purified by flash
chromatography on silica gel (dichloromethane: methanol = 50:1) to yield pure product.
1
Yellow solid (yield 36%). H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 4.9, 1H), 7.45 (d, J =
10.1 Hz, 1H), 7.36–7.33 (m, 2H), 6.36 (d, J = 1.8 Hz, 1H), 4.88–4.82 (m, 1H), 3.94 (s, 3H),
3.69 (d, J = 13.1 Hz, 2H), 3.29–3.22(m, 2H), 2.73 (s, 3H), 2.30 (s, 3H), 2.23 (s, 3H), 2.22 (s,
2H), 1.93–1.89 (m, 2H). HRMS (ESI) calcd for C24H26F4N6O [M+H]+ 491.2177, found
491.2197.
5.1.6. General procedure for the synthesis of compounds 13a–g
N-(1-(6-chloro-4,5-dimethylpyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluoromethyl)
benzamide (10c) (1 equiv), borate ester (2 equiv), potassium fluoride (2 equiv), potassium
phosphate (2 equiv) and m-xylene (5 mL) were added to a microwave vial (20 mL) equipped
with a stir bar. The reaction mixture was purged with nitrogen for 2 min.
Tetrakis(triphenylphosphine)platinum (0.1 equiv) was added and purged with nitrogen for 2
min again. The vial was sealed and irradiated in the microwave at 120 °C (high absorption
setting, Biotage Initiator) for 3 h. The reaction mixture was concentrated under reduced
pressure and dissolved in EtOAc (20 mL), washed with water, dried over magnesium sulfate,

filtered, and concentrated in vacuo. The residue was purified by silica gel column
chromatography (dichloromethane: methanol = 50:1) to yield pure product.
5.1.6.1.
N-(1-(4,5-dimethyl-6-phenylpyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluoromethyl)
benzamide (13a). Yellow solid (yield 31%); 1H NMR (400 MHz, CDCl3) δ 7.55 (dd, J = 8.5,
5.4 Hz, 1H), 7.49–7.45 (m, 3H), 7.43 (d, J = 7.9 Hz, 2H), 7.38 (dd, J = 9.1, 2.8 Hz, 1H), 7.27
(dd, J = 8.8, 3.4 Hz, 1H), 5.95 (d, J = 8.0 Hz, 1H), 4.30–4.15 (m, 1H), 3.59–3.48 (m, 2H),
13
3.22–3.09 (m, 2H), 2.25 (s, 3H), 2.20 (s, 3H), 2.19–2.12 (m, 2H), 1.75–1.71 (m, 2H); C
NMR (151 MHz, CDCl3) δ 166.16, 162.58, 158.08, 138.04, 135.79, 132.30, 131.23 (d, J =
8.2), 129.79, 129.33, 128.20 (d, J = 9.1), 119.15, 119.01, 114.12, 113.99, 49.11, 47.40, 31.70,
16.49, 14.44; HRMS (ESI) calcd for C25H24F4N4O [M+H]+ 473.1959, found 473.1981.
5.1.6.2.
N-(1-(6-(4-chlorophenyl)-4,5-dimethylpyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluoromet
hyl)benzamide (13b). Yellow solid (yield 34%); 1H NMR (400 MHz, CDCl3) δ 7.58 (dd, J =
8.5, 5.4 Hz, 1H), 7.44 (d, J = 1.1 Hz, 4H), 7.41 (dd, J = 8.9, 2.6 Hz, 1H), 7.30 (dd, J = 8.1,
2.5 Hz, 1H), 5.90 (d, J = 8.1 Hz, 1H), 4.34–4.15 (m, 1H), 3.61–3.52 (m, 2H), 3.21–3.14 (m,
2H), 2.28 (s, 3H), 2.22 (s, 3H), 2.18–2.15 (m, 2H), 1.81–1.71 (m, 2H); 13C NMR (151 MHz,
CDCl3) δ 166.15, 162.70, 156.97, 136.49, 135.69, 134.43, 132.27, 131.23 (d, J = 8.3), 130.71,
129.88, 128.46, 119.11 (d, J = 21.2), 114.16, 49.11, 47.40, 31.70, 29.70, 16.45, 14.49; HRMS
(ESI) calcd for C25H23ClF4N4O [M+H]+ 507.1569, found 507.1587.
5.1.6.3.
N-(1-(6-(2-chlorophenyl)-4,5-dimethylpyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluoromet
hyl)benzamide (13c). Yellow solid (yield 33%); 1H NMR (400 MHz, CDCl3). δ 7.59 (dd, J =
8.5, 5.3 Hz, 1H), 7.50–7.45 (m, 1H), 7.43–7.35 (m, 4H), 7.30 (dd, J = 8.2, 2.5 Hz, 1H), 5.95
(d, J = 8.1 Hz, 1H), 4.32–4.18 (m, 1H), 3.62–3.57 (m, 2H), 3.23–3.16 (m, 2H), 2.28 (s, 3H),

13
2.20 (d, J = 12.8 Hz, 2H), 2.06 (s, 3H), 1.80–1.73(m, 2H); C NMR (151 MHz, CDCl3) δ
166.18, 163.09, 156.70, 137.26, 137.07, 135.05, 133.45, 132.30, 131.21 (d, J = 8.3), 129.81,
129.38, 129.23, 128.06, 126.95, 119.16, 119.02, 114.18, 49.03, 47.38, 31.74, 31.67, 24.86,
15.72, 14.35; HRMS (ESI) calcd for C25H23ClF4N4O [M+H]+ 507.1569, found 507.1580.
5.1.6.4.
N-(1-(4,5-dimethyl-6-(p-tolyl)pyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluoromethyl)benz
amide (13d). Yellow solid (yield 32%); 1H NMR (400 MHz, CDCl3) δ 7.58 (dd, J = 8.5, 5.3
Hz, 1H), 7.44–7.36 (m, 3H), 7.32–7.27 (m, 2H), 7.25 (s, 1H), 5.88 (d, J = 8.1 Hz, 1H),
4.33–4.15 (m, 1H), 3.55 (dd, J = 11.0, 6.6 Hz, 2H), 3.21–3.14 (m, 2H), 2.42 (s, 3H), 2.27 (s,
13
3H), 2.23 (s, 3H), 2.21–2.15 (m, 2H), 1.80–1.71 (m, 2H); C NMR (151 MHz, CDCl3) δ
166.14, 162.47, 158.11, 138.04, 135.78, 132.31, 131.24 (d, J = 8.3), 129.72, 129.25, 128.87,
119.18, 119.04, 114.16, 114.00, 49.12, 47.41, 31.73, 21.30, 16.52, 14.42; HRMS (ESI) calcd
for C26H26F4N4O [M+H]+ 487.2116, found 487.2138.
5.1.6.5.
4-fluoro-N-(1-(6-(4-methoxyphenyl)-4,5-dimethylpyridazin-3-yl)piperidin-4-yl)-2-(trifluorom
ethyl)benzamide (13e). White solid (yield 35%); 1H NMR (400 MHz, CDCl3) δ 7.55 (dd, J =
8.5, 5.4 Hz, 1H), 7.45–7.41 (m, 2H), 7.38 (dd, J = 8.9, 2.7 Hz, 1H), 7.24 (dd, J = 8.1, 2.5 Hz,
1H), 7.02–6.95 (m, 2H), 6.16 (d, J = 8.0 Hz, 1H), 4.34–4.13 (m, 1H), 3.86 (s, 3H), 3.54 (d, J
= 13.2 Hz, 2H), 3.24–3.08 (m, 2H), 2.26 (s, 3H), 2.23 (s, 3H), 2.17 (dd, J = 12.9, 3.6 Hz, 2H),
13
1.80–1.71 (m, 2H); C NMR (151 MHz, CDCl3) δ 166.21, 163.36, 162.40, 161.69, 159.67,
157.71, 135.78, 135.05, 132.34, 131.20 (d, J = 8.3), 130.62, 130.52, 130.43, 129.83, 119.06,
118.92, 114.11, 113.95, 113.62, 55.32, 49.14, 47.39, 31.67, 16.55, 14.44; HRMS (ESI) calcd
for C26H26F4N4O2 [M+H]+ 503.2065, found 503.2079.
5.1.6.6.
N-(1-(6-(4-cyanophenyl)-4,5-dimethylpyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluorometh

1
yl)benzamide (13f). White solid (yield 39%); H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.3
Hz, 2H), 7.67–7.63 (m, 2H), 7.59 (dd, J = 8.5, 5.4 Hz, 1H), 7.42 (dd, J = 8.8, 2.5 Hz, 1H),
7.33–7.29 (m, 1H), 5.80 (d, J = 8.0 Hz, 1H), 4.34–4.17 (m, 1H), 3.60 (d, J = 13.3 Hz, 2H),
3.25–3.12 (m, 2H), 2.30 (s, 3H), 2.23 (s, 3H), 2.20 (s, 2H), 1.82–1.69 (m, 2H); 13C NMR
(151 MHz, CDCl3) δ 166.15, 162.94, 156.23, 142.64, 135.60, 132.22, 132.06, 131.25 (d, J =
8.3), 130.14, 129.97, 119.24, 119.10, 118.66, 114.18, 114.02, 112.17, 49.08, 47.40, 31.69,
16.40, 14.58; HRMS (ESI) calcd for C26H23F4N5O [M+H]+ 498.1912, found 498.1931.
5.1.6.7.
N-(1-(4,5-dimethyl-6-(pyridin-4-yl)pyridazin-3-yl)piperidin-4-yl)-4-fluoro-2-(trifluoromethyl)
benzamide (13g). White solid (yield 30%); 1H NMR (400 MHz, CDCl3). δ 8.78–8.67 (m, 2H),
7.59 (dd, J = 8.5, 5.3 Hz, 1H), 7.50–7.44 (m, 2H), 7.42 (dd, J = 8.8, 2.5 Hz, 1H), 7.31–7.27
(m, 1H), 5.82 (d, J = 8.1 Hz, 1H), 4.34–4.16 (m, 1H), 3.61 (d, J = 13.2 Hz, 2H), 3.27–3.11 (m,
2H), 2.30 (s, 3H), 2.25 (s, 3H), 2.24–2.17 (m, 2H), 1.77–1.71 (m, 2H); 13C NMR (151 MHz,
CDCl3) δ 166.15, 163.05, 155.55, 149.82, 145.77, 135.65, 132.23, 131.28, 131.22, 129.91,
124.06, 119.17 (d, J = 21.1), 114.19, 114.02, 49.07, 47.40, 31.69, 16.28, 14.57; HRMS (ESI)
calcd for C24H23F4N5O [M+H]+ 474.1912, found 474.1931.
5.1.7.
tert-butyl(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)carbam
ate (14).
The compound was prepared from tert-butyl (1-(6-chloro-4,5-dimethylpyridazin-3-yl)
piperidin-4-yl)carbamate (8c) using the same general procedure that was used for the
synthesis of compound 11a–c. Light yellow solid (53%); 1H NMR (400 MHz, CDCl3) δ 7.58
(d, J = 1.8 Hz, 1H), 6.37 (d, J = 1.7 Hz, 1H), 4.57 (s, 1H), 3.93 (s, 3H), 3.73 (s, 1H), 3.56 (d,
J = 13.0 Hz, 2H), 3.12 (t, J = 11.6 Hz, 2H), 2.28 (s, 3H), 2.23 (s, 3H), 2.11 (d, J = 11.9 Hz,
2H), 1.68–1.61 (m, 2H), 1.48 (s, 9H); MS (ESI+): [M+H]+: 387.5.

5.1.8.
1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-amine-trifluoroaceta
te (15).
The compound was prepared from tert-butyl(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-
5-yl)pyridazin-3-yl)piperidin-4-yl)carbamate (14) using the same general procedure that was
used for the synthesis of compound 9a–c. Without further purification, the crude material was
used directly into the next reaction.
5.1.9. General procedure for the synthesis of compounds 16a–c
1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-amine-trifluoro
acetate (15) (1 equiv) and ester (1.5 equiv) were added to a microwave vial (20 ml) equipped
with a stir bar. NMP (7 mL) was then added followed by triethylamine (2 ml). The vial was
sealed and irradiated in the microwave at 150 °C (high absorption setting) for 30 min. The
mixture was then diluted with H2O and extracted with EtOAc. The organic layers were dried
over MgSO4 and concentrated. Without further purification, the crude material was used
directly into the next reaction. To a solution of the crude material (1 equiv) in THF (5 mL) at
0 °C was added dropwise CH3MgI (0.86 mL of 3.0 M solution in Et2O, 4 equiv). The reaction
was stirred for 2 h, and then quenched by addition of saturated NH4Cl. The mixture was
extracted with EtOAc and the organic layer was dried over MgSO4, filtered, concentrated,
and then purified by flash chromatography on silica gel (dichloromethane: methanol = 20:1)
to afford the title compound 16a–c.
5.1.9.1.
2-(6-((1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)amino)pyri
1
din-3-yl)propan-2-ol (16a). Light yellow solid (yield 22%); H NMR (400 MHz, CDCl3) δ
8.20 (s, 1H), 7.61 (d, J = 6.9 Hz, 1H), 7.56 (s, 1H), 6.42 (d, J = 8.6 Hz, 1H), 6.36 (s, 1H),
4.64 (s, 1H), 3.92 (s, 4H), 3.60 (d, J = 12.0 Hz, 2H), 3.17 (t, J = 11.4 Hz, 2H), 2.28 (s, 3H),

2.25 (s, 2H), 2.21 (s, 3H), 1.70 (d, J = 10.5 Hz, 2H), 1.56 (s, 6H); 13C NMR (151 MHz,
CDCl3) δ 158.62, 157.32, 149.27, 138.30, 138.05, 137.38, 129.72, 124.80, 115.72, 107.90,
71.63, 49.19, 48.49, 37.92, 32.29, 30.39, 16.08, 14.67; HRMS (ESI) calcd for C23H31N7O
[M+H]+ 422.2663, found 422.2678.
5.1.9.2.
2-(5-((1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)amino)pyra
1
zin-2-yl)propan-2-ol (16b). Light yellow solid (yield 10%); H NMR (400 MHz, CDCl3) δ
7.57 (s, 1H), 7.33 (d, J = 9.3 Hz, 1H), 6.70 (d, J = 9.5 Hz, 1H), 6.36 (s, 1H), 4.68 (s, 1H),
4.17 (s, 1H), 3.93 (s, 3H), 3.63 (d, J = 10.8 Hz, 2H), 3.21 (t, J = 11.6 Hz, 2H), 2.30 (s, 2H),
2.29 (s, 3H), 2.23 (s, 3H), 1.76 (d, J = 10.5 Hz, 2H), 1.56 (s, 6H); 13C NMR (151 MHz,
CDCl3) δ 157.38, 149.26, 138.30, 138.05, 137.38, 129.71, 124.70, 115.69, 107.90, 71.62,
49.19, 48.46, 37.91, 32.30, 30.39, 16.08, 14.67; HRMS (ESI) calcd for C22H30N8O [M+H]+
423.2615, found 423.2611.
5.1.9.3.
2-(6-((1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)amino)pyri
1
dazin-3-yl)propan-2-ol (16c). Light yellow solid (yield 12%); H NMR (400 MHz, CDCl3) δ
7.58 (s, 1H), 7.33 (d, J = 8.9 Hz, 1H), 6.70 (d, J = 9.3 Hz, 1H), 6.37 (s, 1H), 4.57 (s, 1H),
4.22 (s, 1H), 3.93 (s, 3H), 3.64 (d, J = 13.3 Hz, 2H), 3.21 (t, J = 11.5 Hz, 2H), 2.33 (S, 2H),
2.30 (s, 3H), 1.81–1.71 (m, 2H), 1.56 (s, 6H); 13C NMR (151 MHz, CDCl3) δ 162.79, 154.97,
142.42, 140.72, 138.20, 138.10, 137.58, 129.82, 107.95, 48.95, 48.28, 41.44, 37.90, 31.77,
29.69, 16.10, 14.65; HRMS (ESI) calcd for C22H30N8O [M+H]+ 423.2615, found 423.2613.
5.1.10.
N-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)-5-(methylsulf
onyl)pyrazin-2-amine (16d).

1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-amine-trifluoro
acetate (15) (300 mg, 1.05 mmol) and 2-bromo-5-(methylsulfonyl)pyrazine (360 mg, 1.5
mmol) were added to a microwave vial (20 ml) equipped with a stir bar. NMP (7 mL) was
then added followed by triethylamine (2 ml). The vial was sealed and irradiated in the
microwave at 150 °C (high absorption setting) for 30 min. The mixture was then diluted with
H2O and extracted with EtOAc. The organic layers were dried over MgSO4, filtered, and
concentrated. The residue was purified by flash chromatography on silica gel
(dichloromethane: methanol = 10:1) to yield pure product. Light yellow solid (yield 18%); 1H
NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 7.93 (s, 1H), 7.56 (s, 1H), 6.36 (s, 1H), 5.72 (d, J =
6.6 Hz, 1H), 4.17 (s, 1H), 3.90 (s, 3H), 3.62 (d, J = 12.7 Hz, 2H), 3.20 (t, J = 11.4 Hz, 2H),
13
3.13 (s, 3H), 2.29 (s, 3H), 2.22 (s, 3H), 2.18 (s, 2H), 1.76 (d, J = 10.6 Hz, 2H); C NMR
(151 MHz, CDCl3) δ 152.69, 149.53, 138.59, 138.05, 137.52, 137.00, 129.83, 125.48, 107.72,
71.02, 56.33, 51.26, 48.57, 37.78, 31.93, 30.40, 30.37, 29.70, 29.36, 15.95, 15.47; HRMS
(ESI) calcd for C20H26N8O2S [M+H]+ 443.1972, found 443.1993.
5.1.11. General procedure for the synthesis of compounds 17a–d
Amine (1 equiv) was dissolve in dichloromethane (10 mL). The obtained solution was
cooled down on an ice-water bath. Then phenyl carbonochloridate (1.2 equiv), pyridine (1.2
equiv) were added in order. The reaction mixture was stirred for 5 h while warming at room
temperature, and was concentrated afterwards. Without further purification, the crude
material was used directly into the next reaction.
5.1.11. General procedure for the synthesis of compounds 18a–d
1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-amine-trifluoro
acetate (15) (1.6 equiv), carbamate (1 equiv) and triethylamine (2 equiv) were added to
DMSO (5 mL) under nitrogen. The mixture was then stirred at 100 °C for 8 h. The reaction
solution was washed with water and extracted with EtOAc, dried over magnesium sulfate,

filtered, and concentrated in vacuo. The residue was purified by silica gel column
chromatography (dichloromethane: methanol = 20:1) to yield pure product.
5.1.11.1.
1-cyclopentyl-3-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)
piperidin-4-yl)urea(18a). White solid (yield 20%); 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J =
1.7 Hz, 1H), 6.35 (d, J = 1.7 Hz, 1H), 4.63 (s, 2H), 3.96 (dd, J = 13.3, 6.5 Hz, 1H), 3.91 (s,
3H), 3.54 (d, J = 12.9 Hz, 2H), 3.13 (t, J = 11.3 Hz, 2H), 2.27 (s, 3H), 2.21 (s, 3H), 2.09 (d, J
13
= 10.7 Hz, 2H), 1.97–1.93 (m, 4H), 1.66–1.56 (m, 4H), 1.39–1.36 (m, 2H); C NMR (151
MHz, CDCl3) δ 162.94, 157.18, 149.12, 138.31, 138.04, 137.33, 129.66, 107.89, 52.26, 49.17,
47.11, 37.90, 33.66, 32.91, 23.62, 16.06, 14.68; HRMS (ESI) calcd for C21H31N7O [M+H]+
398.2663, found 398.2663.
5.1.11.2.
1-cycloheptyl-3-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)
piperidin-4-yl)urea (18b). White solid (yield 25%); 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J
= 1.9 Hz, 1H), 6.42 (s, 1H), 3.90 (s, 3H), 3.82 (s, 1H), 3.65 (d, J = 14.1 Hz, 3H), 3.21 (t, J =
11.9 Hz, 2H), 2.33 (s, 3H), 2.26 (s, 3H), 2.11 (d, J = 12.6 Hz, 2H), 1.95 (d, J = 13.8 Hz, 2H),
13
1.76–1.68 (m, 2H), 1.65–1.59 (m, 4H), 1.54–1.46 (m, 6H). C NMR (151 MHz, CDCl3) δ
162.94, 156.57, 149.13, 138.04, 129.65, 107.89, 51.52, 49.18, 47.21, 41.04, 37.90, 35.75,
32.92, 28.11, 24.08, 16.05, 14.67; HRMS (ESI) calcd for C23H35N7O [M+H]+ 426.2976,
found 426.2976.
5.1.11.3.
1-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)-3-phenylurea
1
(18c). Yellow solid (yield 28%); H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.28 (s, 3H),
7.03 (s, 2H), 6.35 (s, 1H), 5.26 (d, J = 6.7 Hz, 1H), 3.97 (s, 1H), 3.88 (s, 3H), 3.51 (d, J =
13.3 Hz, 2H), 3.13 (t, J = 11.6 Hz, 2H), 2.63 (s, 1H), 2.25 (s, 3H), 2.20 (s, 3H), 2.06 (s, 2H),
1.61 (d, J = 9.8 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ 155.17, 138.75, 138.19 (d, J = 19.6),

137.59, 129.93, 129.25, 123.62, 120.74, 107.94, 48.93, 46.89, 37.84, 32.44, 16.07, 14.75;
HRMS (ESI) calcd for C22H27N7O [M+H]+ 406.2350, found 406.2368.
5.1.11.4.
1-(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)-3-(pyridin-3-y
1
l)urea (18d). Yellow solid (yield 22%); H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H), 8.44 (s,
1H), 8.26–8.14 (m, 2H), 7.54 (d, J = 1.7 Hz, 1H), 6.37 (s, 1H), 6.10 (d, J = 6.8 Hz, 1H), 3.98
(s, 1H), 3.86 (s, 3H), 3.45 (s, 2H), 3.13 (dd, J = 13.3, 8.6 Hz, 2H), 2.29 (s, 3H), 2.20 (s, 3H),
13
1.60 (d, J = 8.8 Hz, 2H), 1.39 (dd, J = 16.2, 8.8 Hz, 2H); C NMR (151 MHz, CDCl3) δ
163.41, 154.98, 149.25, 142.61, 139.83, 138.25, 138.10, 137.24, 130.80, 126.80, 124.11,
108.03, 48.39, 46.07, 37.77, 31.83, 16.08, 14.72, 8.68; HRMS (ESI) calcd for C21H26N8O
[M+H]+ 407.2302, found 407.2319.
5.1.12. Phenyl
(1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-yl)carbamate (19).
1-(4,5-dimethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridazin-3-yl)piperidin-4-amine-trifluoro
acetate (15) (100 mg, 0.25 mmol) was dissolved in dichloromethane (10 mL) and charged
with pyridine (200 mg, 2.5 mmol) in drops. The mixture was stirred at room temperature for
10 min, then phenyl carbonochloridate (46 mg, 0.3 mmol) was added. The obtained solution
was stirred at room temperature for 6 h. The mixture was then diluted with H2O and extracted
with methylene chloride. The organic layers were dried over MgSO4 and concentrated. The
residue was purified by flash chromatography on silica gel (dichloromethane: methanol =
20:1) to afford the title compound 19. Yellow solid (yield 20%); 1H NMR (400 MHz, CDCl3)
δ 7.57 (s, 1H), 7.36 (t, J = 7.7 Hz, 2H), 7.20 (t, J = 7.5 Hz, 1H), 7.14 (d, J = 7.9 Hz, 2H), 6.36
(s, 1H), 5.08 (d, J = 7.8 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 1H), 3.59 (d, J = 13.3 Hz, 2H), 3.15 (t,
J = 11.5 Hz, 2H), 2.28 (s, 3H), 2.22 (s, 3H), 2.18 (s, 2H), 1.75 (dd, J = 20.4, 10.5 Hz, 2H);
13C NMR (151 MHz, CDCl3) δ 162.83, 153.75, 149.31, 138.24, 138.04, 137.41, 129.73,