Towards dual inhibitors of the MET kinase and WNT signaling pathway; Design, synthesis and biological evaluation

Article abstract of DOI:10.1039/c9ra08954c

Both the kinase MET and the WNT signaling pathway are attractive targets in cancer therapy, and synergistic effects have previously been observed in animal models upon simultaneous inhibition. A strategy towards a designed multiple ligand of MET and WNT signaling is pursued based on the two hetero biaryl systems present in both the MET inhibitor tepotinib and WNT signaling inhibitor TC-E 5001. Initial screening was conducted to find the most suitable ring systems for further optimization, whereas a second screen explored modifications towards pyridazinones and triazolo pyridazines. Up to 54% reduction of WNT signaling activity at 10 μM concentration was achieved, however, only low affinities towards MET were observed. Overall, the thiophene substituted pyridazinone 40 was the best dual MET and WNT signaling inhibitor, with a 17% and 19% reduction of activity, respectively. Although further optimizations are needed to achieve more potent dual inhibitors, the strategy presented herein can be valuable towards the development of a dual inhibitor of MET and WNT signaling.

Full text of DOI:10.1039/c9ra08954c

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PAPER
Towards dual inhibitors of the MET kinase and WNT
signaling pathway; design, synthesis and biological
evaluation†
Cite this: RSC Adv., 2019, 9, 37092
a
Margrethe Konstanse Kristiansen,a Solveig Pettersen,b
*
Vegard Torp Lien,
Mads Haugland Haugen,b Dag Erlend Olberg,ac Jo Waalerd and Jo Klavenessa
Both the kinase MET and the WNT signaling pathway are attractive targets in cancer therapy, and synergistic
effects have previously been observed in animal models upon simultaneous inhibition. A strategy towards
a designed multiple ligand of MET and WNT signaling is pursued based on the two hetero biaryl systems
present in both the MET inhibitor tepotinib and WNT signaling inhibitor TC-E 5001. Initial screening was
conducted to find the most suitable ring systems for further optimization, whereas a second screen
explored modifications towards pyridazinones and triazolo pyridazines. Up to 54% reduction of WNT
signaling activity at 10 mM concentration was achieved, however, only low affinities towards MET were
observed. Overall, the thiophene substituted pyridazinone 40 was the best dual MET and WNT signaling
inhibitor, with a 17% and 19% reduction of activity, respectively. Although further optimizations are
needed to achieve more potent dual inhibitors, the strategy presented herein can be valuable towards
the development of a dual inhibitor of MET and WNT signaling.
Received 12th September 2019
Accepted 4th November 2019
DOI: 10.1039/c9ra08954c
rsc.li/rsc-advances
developed, which synergistically reduced tumor growth in
a lung cancer model in vivo.11
Introduction
While the prevalent strategy in drug discovery is “one drug,
one target”, the realization that many drugs exerts their effect
through more than one target have led to the increased interest
in designed multiple ligands (DML).12,13 Well-designed DMLs
can reduce drug–drug interactions and side effects, and
simplify drug pharmacokinetics.14,15 In search of novel treat-
ment strategies for cancer, we aim to develop dual inhibitors of
MET and WNT signaling.
The proteins tankyrase 1 and 2 (TNKS1/2) are central regu-
lators of WNT signaling,16 and TNKS1/2 inhibitors are prom-
ising in the development of WNT signaling modulators. TNKS1/
2 poly(ADP-ribosyl)ate AXIN proteins to earmark them for
degradation by the ubiquitin–proteasomal system. Inhibition of
TNKS1/2 can therefore lead to stabilization of AXIN proteins
and hence the b-catenin degradosomes that in turn enhances
the degradation of b-catenin.17 The TNKS1/2 inhibitors can
attain three different binding modes in the active site. This,
combined with the fact that many of the known inhibitors are
discovered through high-throughput screening leads to struc-
tures spanning a wide chemical space.18 Some examples of
TNKS1/219–21 and MET inhibitors22–26 are shown in Fig. 1.
The selective MET inhibitors have traditionally two fused
heterocycles connected with a one or two atom linker, as
exemplied with structures 4–7. Several different heterocyclic
moieties have been explored, and the linker enables the ligands
to adopt a U-shape in the active site of MET.27 One example of
a MET inhibitor deviating from this pattern is tepotinib (8),
The tyrosine kinase MET and the WNT/b-catenin signaling
pathway (WNT signaling) have both received considerable
interest as targets in cancer therapy due their involvement in
carcinogenesis.1–3 MET is dysregulated in a wide range of
malignancies, and is correlated with a poor prognosis.4 WNT
signaling is critical for cell repair and stem cell maintenance,
and aberrant activity is closely associated with the development
and growth of a plethora of cancerous diseases. Therefore,
small-molecule WNT signaling inhibitors are predicted to be
effective anti-tumor drugs in humans.5 Importantly, both MET
and WNT signaling pathways hold a central role in the devel-
opment of metastasis, and a strong biochemical connection
between the two have been documented.6,7 Hence, simulta-
neous inhibition have demonstrated synergistic effects in
breast cancer models,8 and overcoming of resistance in lung
cancer9 and melanoma cell lines.10 In addition, a bispecic
antibody inhibiting both MET and WNT signaling has been
aDepartment of Pharmacy, University of Oslo, Oslo, Norway. E-mail: v.t.lien@farmasi.
uio.no
bDepartment of Tumor Biology, Institute for Cancer Research, OUS Radiumhospitalet,
Oslo, Norway
cNorsk Medisinsk Syklotronsenter AS, OUS Rikshospitalet, Oslo, Norway
dDepartment of Microbiology, Section for Cell Signaling, OUS Rikshospitalet, Oslo,
Norway
† Electronic supplementary information (ESI) available: Full synthetic and
analytical details for all compounds are given in the ESI. See DOI:
10.1039/c9ra08954c
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Fig. 1 Examples of inhibitors of TNKS1/2 and MET.
consisting of two unfused hetero biaryl systems. As such, 15 is similar to tepotinib (8), while pyrazolo pyridine 16 is
tepotinib creates a link from the traditional MET inhibitors to inspired by the MET inhibitor 6.
the TNKS1/2 inhibitor TC-E 5001 (2). This similarity is elabo-
rated in Fig. 2a; two hetero biaryls, each colored in red or blue,
connected by a linker make up a common structural core. For
Results and discussion
a dual MET and WNT signaling inhibitor, these two pharma-
To evaluate the hypothesis that known MET and TNKS1/2
cophores are merged to the target scaffold 9 in Fig. 2b. In 9, the
inhibitors have structural similarities that can be applied in
ring systems can be either fused or unfused, and the positions
the development of dual MET and WNT signaling inhibitors, in
of heteroatoms and substituents can be altered for structural
silico docking was performed. Tepotinib and TC-E 5001 were
optimization.
docked into the TNKS1 and MET active site, respectively, and
Initially, the aim was to evaluate which heteroaromatic
a good overlap with the experimental structures were observed
ring systems that best could be applied in the target structure
in both proteins (Fig. 3). The pyridazinone in tepotinib and the
9. By utilizing a SN2 disconnection and commercial starting
triazole in TC-E 5001 replace each other in the two active sites,
materials, a selection of structural moieties from known
and the linkers enables the molecules to adopt the conforma-
TNKS1/2 and MET inhibitors could be combined efficiently to
tions observed in both parent crystallographic structures.
the target scaffold 9. In Fig. 2c, oxadiazole 10 is similar to the
Based on the target scaffold 9 in Fig. 2b, combinations of the
oxadiazole in TC-E 5001 (2), while 11 represents a fused
nucleophiles and electrophiles in Fig. 2c resulted in the mole-
analog of this moiety, and at the same time bearing simi-
cules presented in Fig. 4, which were synthesized and evaluated
larities with MET inhibitors 6 and 7. Phthalimide 12 is
in enzymatic MET (Cyclex) and cellular luciferase-based WNT/b-
inspired by the TNKS1/2 inhibitors WIKI4 (1) and IWR-1 (3).
catenin signaling reporter assays (SuperTOPFlash assay) at 10
Triazole 13 is inspired by the triazole in TC-E 5001 (2), while
mM concentration.
the triazolopyridine 14 is the fused analog of 13, at the same
The triazolopyridine 18 was the most potent in the WNT
time representing known MET inhibitors. The pyridazinone
signaling assay with a 50% reduction of activity, while the
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Fig. 2 Construction of the generalized target structure 9. (a) Similarities between TC-E 5001 (2) and tepotinib (8) are highlighted; (b) merging of
pharmacophores yields the target structure 9; (c) the target structure can be synthesized via SN2 reactions from commercial starting materials.
See the experimental data for synthetic details.
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unfused analog 17 was inactive. The pyridazinones displayed
the same inhibition (25%) regardless of whether an unfused
oxadiazole (21) or a fused oxazolo pyridine (22) was present. The
large structural similarity between 19 and TC-E 5001 (2) and the
modest inhibition of WNT signaling activity with 19 should be
noted, indicating a relatively sensitive pharmacophore. MET
inhibition did not reach the anticipated level for neither of the
analyzed compounds.
With two fused heterocycles, 18 shares common chemical
characteristics with known MET inhibitors, and should allow
for improvement of the MET affinity. The pyridazinone in 21
and 22 can also serve as a foundation for optimization, and
further modications were therefore based on 18, 21 and 22.
With the syntheses shown in Scheme 1, the strategy was to
interchange the triazolo pyridine in 18 to a triazolo pyridazine,
Fig. 3 In silico docking of: (a) tepotinib (8) and TC-E 5001 (2) in MET;
(b) tepotinib (8) and TC-E 5001 (2) in TNKS1.
Fig. 4 Molecules evaluated in the initial screen at 10 mM concentration, presented with values from MET and WNT signaling assays. Inhibition
values are presented as averages from $2 parallels.
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Scheme 1 Synthesis of pyridazinone and triazolo pyridazine analogs. (i) Boronic acid, Pd(dppf)Cl2$CH2Cl2, K2CO3, 1,4-dioxane, H2O, 80 ꢀC, 15 h;
(ii) hydrazine hydrate, EtOH, 80 ꢀC, 3–5 h; (iii) CS2, KOH, EtOH, rt for 48 h or 80 ꢀC for 15 h; (iv) AcOH, 80 ꢀC for 4 h or 100 ꢀC for 15 h. Yields are
given for isolated structures.
and to introduce various substituents on the pyridazinone and
triazolo pyridazine.
Conclusion
Both pyridazinones and triazolo pyridazines could be
synthesized starting from 3,6-dichloropyridazine (28). Suzuki–
Miyaura coupling gave the intermediate chloro pyridazines 29,
which could be hydrolyzed with AcOH to substituted pyr-
idazinones 32. Nucleophilic attack on 29 by hydrazine hydrate
and subsequent cyclization with CS2 gave the corresponding
triazolo pyridazines 31. To obtain the target structures, the
pyridazinones and triazolo pyridazines were reacted with elec-
trophilic chlorides. To potentially increase the affinity for MET,
the oxadiazole pyrimidine 33 was included due to reported
interactions between the pyrimidine ring and the active site in
the crystal structure of tepotinib in MET.26 The novel analogs
were evaluated in MET and WNT signaling assays, and the
results are presented in Fig. 5.
Overall, the performed modications did not increase the
affinity for MET. Comparing e.g. 35 or 38 with tepotinib, the
main difference is the oxadiazole in place for the phenyl group.
This modication can alter the orientation of the ligand,
although successful modications have been made at this
position earlier.28,29 Neither of the triazolo pyridazines 44 and 45
were able to inhibit MET, despite large similarities with e.g.
AMG-208 (4). An increase from 25% to 54% in WNT signaling
inhibition was obtained when moving from 21 and 22 to 35,
however, low WNT signaling inhibition was observed for the
remaining pyridazinones. Optimization of 18 to the structures
44 and 45 led to a similar or somewhat reduced WNT signaling
inhibition. For this triazolo pyridazine series, the chlorine
substituent in 42 and 43 reduced the WNT inhibition. The best
combined inhibition of MET and WNT signaling, albeit modest,
was achieved with the thiophene analog 40, with 17% and 19%
reduction, respectively.
The design of a scaffold for potential dual inhibitors of MET
and WNT signaling was performed from the MET inhibitor
tepotinib (8) and the tankyrase inhibitor TC-E 5001 (2). The
target scaffold 9 was designed so that facile SN2 reactions would
furnish the nal products directly, facilitating screening of the
most favorable structural cores. In silico docking revealed
a potential good overlap of tepotinib and TC-E 5001 in both
active sites. The rst compound screen demonstrated low MET
inhibition, while WNT signaling activity could be reduced by
50% at 10 mM concentration. Further structural modications
were carried out based on the triazolo pyridine 18 and pyr-
idazinones 21 and 22. In the second compound screen, the
pyridazinone 35 inhibited WNT signaling by 54%, however with
no inhibition of MET. The thiophene substituted pyridazinone
40 was overall the best dual MET and WNT signaling inhibitor,
with a 17% and 19% reduction of activity, respectively. Although
further structural optimizations are needed, the novel strategy
provided herein could prove valuable for further development
of dual MET and WNT signaling inhibitors.
Experimental
General
All chemicals were purchased from Sigma-Aldrich or Fluorochem
and used without further purication. Air and/or moisture sensi-
tive reactions were performed under argon atmosphere with dried
solvents and reagents. TLC was performed on Merck silica gel 60
F254 plates, and visualized using UV light at 312 nm or 365 nm,
a phosphomolybdic acid solution (12 g phosphomolybdic acid in
250 mL EtOH) or a potassium permanganate (1.5 g KMnO4, 10 g
K2CO3, 2.5 mL 5 M NaOH/H2O, 200 mL H2O) solution for detec-
tion. Column chromatography was performed with silica gel (pore
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Fig. 5 Molecules evaluated in the second screen at 10 mM concentration, presented with values from MET and WNT signaling assays. Inhibition
values are presented as averages from $2 parallels.
1
size 60 A, 230–400 mesh particle size). H and 13C NMR spectra
ꢀ
General procedure for the alkylations towards the biologically
were obtained on a Bruker AVIII HD 400 instrument (400/101
MHz) or Bruker AVII 600 (600/151 MHz). Chemical shis (d) are
reported in parts per million (ppm), and coupling constants are
reported in hertz (Hz). The residual proton solvent resonance in 1H
NMR (CDCl3 at d 7.27, DMSO-d6 at d 2.50) and the residual carbon
solvent resonance in 13C NMR (CDCl3 at d 77.16 ppm and DMSO-d6
at d 39.52) are used as reference. Accurate mass determination
(HRMS) in positive or negative mode was performed on a Waters
Prospec Q instrument, ionized by electrospray (ESI). LC-MS was
performed on a Thermo Finnigan LCQ Deca XP Plus with
a gradient from 10% to 90% acetonitrile in water over 10 minutes.
tested compounds
The nucleophile and Cs2CO3 were dissolved in DMF (2 mL).
Aer 5 minutes, the electrophile dissolved in DMF (1 mL) was
added dropwise, and the reaction was stirred at ambient
temperature for 15 hours (70 ꢀC where noted). Work-up and
purication: method A; The reaction mixture was diluted with
EtOAc (2 mL) and water (2 mL), and the organic phase was
separated. The aqueous phase was extracted with EtOAc (2 ꢁ 5
mL), and the combined organic phases were washed with water
(4 ꢁ 5 mL) and brine (5 mL), dried over MgSO4 and reduced on
a rotary evaporator. The crude product was puried by column
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chromatography (Hep : EtOAc (1 : 1) / Hep : EtOAc : MeOH DMSO-d6) d 163.1 (d, J ¼ 243.5 Hz), 160.7, 143.1 (d, J ¼ 2.94 Hz),
(10 : 10 : 2)). Method B: The reaction mixture was reduced on 137.5 (d, J ¼ 7.98 Hz), 131.9, 131.5 (d, J ¼ 8.38 Hz), 130.6, 122.2
a rotary evaporator, and puried by column chromatography (2– (d, J ¼ 2.74 Hz), 116.4 (d, J ¼ 20.83 Hz), 112.8 (d, J ¼ 23.31 Hz).
5% MeOH in DCM).
6-(Pyridin-4-yl)pyridazin-3(2H)-one (32b). AcOH (3 mL) was
added to 3-chloro-6-(pyridin-4-yl)pyridazine (29b) (100 mg, 0.52
5-(([1,2,4]Triazolo[4,3-a]pyridin-3-ylthio)methyl)-3-(p-tolyl)-
1,2,4-oxadiazole (17). Prepared according to the general proce- mmol) and stirred at 100 ꢀC for 15 hours. The reaction mixture
dure by reacting [1,2,4]triazolo[4,3-a]pyridine-3-thiol (14) was reduced on a rotary evaporator, and the title compound was
(40 mg, 0.265 mmol) and 5-(chloromethyl)-3-(p-tolyl)-1,2,4- obtained as a grey solid (82 mg, 91%), and used in the next steps
oxadiazole (10) (71 mg, 0.340 mmol) in the presence of without further purication. The spectroscopic analysis was in
Cs2CO3 (85 mg, 0.261 mmol). Work-up and purication were agreement with earlier reported data.28 1H NMR (400 MHz,
performed according to method A and the title compound was DMSO-d6) d 13.81 (bs, 1H), 8.94 (d, 2H, J ¼ 6.5 Hz), 8.37 (d, 2H, J
obtained as a white solid (58 mg, 67%). 1H NMR (400 MHz, ¼ 6.5 Hz), 8.28 (d, 1H, J ¼ 9.9 Hz), 7.12 (d, 1H, J ¼ 9.9 Hz).
CDCl3): d 8.10 (d, 1H, J ¼ 6.9 Hz), 7.81 (d, 1H, J ¼ 9.2 Hz), 7.73 (d,
6-(5-Acetylthiophen-2-yl)pyridazin-3(2H)-one
(32c).
3,6-
2H, J ¼ 8.2 Hz), 7-32-7-28 (m, 1H), 7.22 (d, 2H, J ¼ 8.0), 6.85 (t, Dichloropyridazine (28) (301 mg 2.02 mmol), (5-acetylthiophen-
1H, J ¼ 6.7 Hz), 4.45 (s, 2H), 2.39 (s, 3H). 13C NMR (101 MHz, 2-yl)boronic acid (339 mg, 1.99 mmol), K2CO3 (592 mg, 4.28
CDCl3) d 175.27, 168.77, 151.60, 141.95, 138.64, 129.63, 128.18, mmol) and Pd(dppf)Cl2$CH2Cl2 (164 mg, 0.201 mmol) in 1,4-
127.36, 123.35, 123.23, 116.69, 114.61, 30.28, 21.67. HRMS dioxane (10 mL) and H2O (3 mL) were reacted according to the
(ESI+) m/z calcd for C16H13N5NaOS [MNa]+: 346.0733, found general protocol. The intermediate 1-(5-(6-chloropyridazin-3-yl)
346.0733.
thiophen-2-yl)ethan-1-one (29c) was hydrolyzed in AcOH (10
mL). The title compound was obtained as a brown solid (69 mg,
16%). 1H NMR (400 MHz, DMSO-d6) d 13.26 (s, 1H), 8.09 (d, J ¼
Synthesis of intermediates
General procedure for Suzuki–Miyaura reactions. 3,6- 9.86 Hz, 1H), 7.95 (d, J ¼ 3.82 Hz, 1H), 7.78 (d, J ¼ 3.69 Hz, 1H),
Dichloropyridazine, boronic acid and K2CO3 were put under 7.02 (d, J ¼ 10.2 Hz, 1H), 2.54 (s, 3H). 13C NMR (400 MHz,
argon atmosphere. 1,4-dioxane and H2O were added using DMSO-d6) d 191.5, 160.5, 146.8, 144.8, 140.3, 135.1, 131.0, 130.7,
syringes, and the solution was degassed with argon for 15 127.8, 26.95. HRMS (ESI+) m/z calcd for C10H8N2O2S [MNa]+:
minutes. Pd(dppf)Cl2$CH2Cl2 was added, the solution degassed 243.0199, found 243.0198.
for 5 minutes and the reaction tube was sealed. The reaction
6-(2,4-Diuorophenyl)pyridazin-3(2H)-one (32d). 3,6-Dichlor-
mixture was stirred at 80 ꢀC for 15 hours and reduced on opyridazine (28) (54 mg, 0.36 mmol), 2,4-diuorophenyl
a rotary evaporator. AcOH was added and the mixture was boronic acid (68 mg, 0.43 mmol), K2CO3 (99 mg, 0.72 mmol)
ꢀ
stirred at 80 C for 4 hours (except 32b). The reaction mixture and Pd(dppf)Cl2$CH2Cl2 (24 mg, 0.029 mmol) in 1,4-dioxane (2
was reduced on a rotary evaporator and the crude product was mL) and H2O (0.5 mL) were reacted according to the general
puried by column chromatography (5% MeOH in DCM).
protocol. The intermediate 3-chloro-6-(2,4-diuorophenyl)
3-Chloro-6-(pyridin-4-yl)pyridazine (29b). 3,6-Dichloropyr- pyridazine (29d) was hydrolyzed in AcOH (3 mL). The title
1
idazine (28) (686 mg, 4.6 mmol), 4-pyridinyl boronic acid compound was obtained as a light grey solid (30 mg, 40%). H
(531 mg, 4.3 mmol), K2CO3 (565 mg, 4.1 mmol) and Pd(dppf) NMR (600 MHz, DMSO-d6) d 13.35 (s, 1H), 7.76–7.70 (m, 2H),
Cl2$CH2Cl2 (170 mg, 0.208 mmol) in 1,4-dioxane (6 mL) and H20 7.42 (ddd, 1H, J ¼ 11.3 Hz, 9.0 Hz, 2.5 Hz), 7.23 (td, 2H, J ¼ 8.5,
(2 mL) were reacted according to the general protocol. The 2.4 Hz), 6.99 (d, 1H, J ¼ 9.90 Hz). 13C NMR (151 MHz, DMSO-d6)
reaction mixture was reduced on a rotary evaporator and the d 162.7 (dd, J ¼ 249.3, 12.4 Hz), 160.0, 159.6 (dd, J ¼ 251.2, 12.6
crude product was puried by column chromatography (5% Hz), 140.3 (d, J ¼ 1.7 Hz), 133.8 (d, J ¼ 5.7 Hz), 131.3 (dd, J ¼
MeOH in DCM) and the title compound was obtained as a grey 10.0, 4.2 Hz), 129.8, 119.9 (dd, J ¼ 12.3, 3.7 Hz), 112.3 (dd, J ¼
solid (349 mg, 42%). The spectroscopic analysis was in agree- 21.5, 3.5 Hz), 104.8 (t, J ¼ 26.2 Hz). HRMS (ESI+) m/z calcd for
ment with earlier reported data.28 1H NMR (400 MHz, CDCl3)
d 8.83 (d, 2H, J ¼ 5.5 Hz), 7.98 (d, 2H, J ¼ 5.7 Hz), 7.90 (d, 1H, J ¼
8.9 Hz), 7.66 (d, 1H, J ¼ 8.9 Hz).
C
10H6F2N2NaO [M + H]+: 231.0340, found 231.0340.
6-Chloro-[1,2,4]triazolo[4,3-b]pyridazine-3-thiol (31a). 3,6-
Dichloropyridazine (28) (214 mg, 1.44 mmol) was suspended in
6-(3-Fluorophenyl)pyridazin-3(2H)-one (32a). 3,6-Dichloropyr- EtOH (3 mL) and added hydrazine hydrate (74 mL, 1.53 mmol).
idazine (28) (249 mg, 1.68 mmol), (3-uorophenyl)boronic acid The resultant mixture was heated to reux for 3 hours and
24 (286 mg, 2.04 mmol), K2CO3 (460 mg, 3.32 mmol) and reduced on a rotary evaporator. The intermediate 3-chloro-6-
Pd(dppf)Cl2$CH2Cl2 (136 mg, 0.167 mmol) in 1,4-dioxane (10 hydrazinylpyridazine (30a) (36 mg, 0.249 mmol) was sus-
mL) and H2O (3 mL) were reacted according to the general pended in EtOH (1.5 mL) and added a solution of KOH (14 mg,
protocol.
The
intermediate
3-chloro-6-(3-uorophenyl) 0.249 mmol) in H2O (1.5 mL). CS2 (30 mL, 0.496 mmol) was
pyridazine (29a) was hydrolyzed in AcOH (3 mL). The title added and the mixture was stirred at ambient temperature for
compound was obtained as a light grey solid (153 mg, 48%). The 48 h, with similar addition of KOH and CS2. The reaction
spectroscopic analysis was in agreement with earlier reported mixture was reduced on a rotary evaporator and puried by
data.30 1H NMR (400 MHz, DMSO-d6) d 13.27 (s, 1H), 8.06 (d, J ¼ column chromatography (5–10% MeOH in DCM). The title
9.93 Hz, 1H), 7.71 (d, J ¼ 8.02 Hz, 1H), 7.67 (dt, J ¼ 10.48, compound was obtained as a yellow solid (7 mg, 15% over two
2.08 Hz, 1H), 7.52 (td, J ¼ 8.16 Hz, 6.17 Hz, 1H), 7.27 (td, J ¼ steps). The spectroscopic analysis was in agreement with earlier
8.52, 2.66 Hz, 1H), 7.00 (d, J ¼ 9.94 Hz, 1H). 13C NMR (400 MHz, reported data.31 1H NMR (600 MHz, DMSO-d6) d 14.92 (s, 1H),
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8.24 (d, 2H, J ¼ 9.77 Hz), 7.48 (d, 2H, J ¼ 9.77 Hz). 13C NMR (151
MHz, DMSO-d6) d 160.47, 148.74, 140.16, 126.67, 124.75. HRMS
(ESIꢂ) m/z calcd for C5H2ClN4S [M ꢂ H]ꢂ: 184.9694, found
184.9694.
Conflicts of interest
There are no conicts to declare.
6-(Pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazine-3-thiol (31b). 3-
Chloro-6-(pyridin-4-yl)pyridazine (29b) (250 mg, 1.30 mmol) was
suspended in EtOH (4 mL) and added hydrazine hydrate (150
mL, 3.1 mmol). The resultant mixture was heated to reux for 5
hours in a sealed tube and reduced on a rotary evaporator. The
intermediate 3-hydrazinyl-6-(pyridin-4-yl)pyridazine (30b) was
suspended in EtOH (5 mL) and added KOH (90 mg, 1.61 mmol).
The reaction mixture was placed under argon and degassed for
5 minutes. CS2 (30 mL, 0.496 mmol) was added and the mixture
was stirred in a closed vial at 80 ꢀC for 15 h. The reaction
mixture was reduced on a rotary evaporator and puried by
column chromatography (5–10% MeOH in DCM). The title
compound was achieved as an orange solid (92 mg, 31% over
two steps). 1H NMR (400 MHz, DMSO-d6) d 14.89 (bs, 1H), 8.82
(m, 2H), 8.32 (d, 1H, J ¼ 10.0 Hz), 8.06 (m, 2H), 8.01 (d, 1H, J ¼
10.90 Hz). 13C NMR (101 MHz, DMSO-d6) d 151.38, 150.67,
141.71, 141.06, 126.16, 121.96, 121.08. HRMS (ESI+) m/z calcd
for C10H8N5S [M + H]+: 230.0495, found 230.0495.
Notes and references
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ꢀ
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