Synthesis and antitumor activity of novel pyridazinone derivatives containing 1,3,4-thiadiazole moiety

Article abstract of DOI:10.1080/10426507.2020.1737062

A series of novel pyridazinone derivatives containing the 1,3,4-thiadiazole moiety were synthesized and characterized by ¹H NMR, 13C NMR, spectroscopies HRMS and IR. Among them, the structure of compound 5c (2-(Tert-butyl)?4-chloro-5-((5-((2-ethylphenyl)amino)?1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One) was unambiguously confirmed via single crystal X-ray diffraction analysis. The inhibitory activity of all the target compounds against MGC-803 and Bcap-37 was determined by MTT assay, with doxorubicin (the inhibition rates were 95.5 ± 0.4% and 95.7 ± 1.0% respectively) as a control. The preliminary results showed that the inhibitory activity of compound 5n (2-(Tert-butyl)?4-chloro-5-((5-((3-fluorophenyl)amino)?1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One) was superior to the others. The inhibition rates of MGC-803 and Bcap-37 cells were 86.3 ± 2.2% and 92.3 ± 0.6% at a concentration of 10 μmol/L, respectively. The preliminary structure-activity relationship showed that when the 2-position of the benzene ring was substituted by a methyl group, such as compound 5j (2-(Tert-butyl)?4-chloro-5-((5-((2,3-dimethylphenyl)amino)?1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One), it exhibited good anticancer activity on MGC-803 cells. Besides, introducing fluorine, chlorine, or trifluoromethyl group onto the benzene ring, such as compound 5 m (2-(Tert-butyl)?4-chloro-5-((5-((4-(trifluoromethoxy)phenyl)amino)?1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One), displayed good anticancer activity on MGC-803 and Bcap-37 cells.

Full text of DOI:10.1080/10426507.2020.1737062

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Synthesis and antitumor activity of novel
pyridazinone derivatives containing 1,3,4-
thiadiazole moiety
Junhu Qin, Mei Zhu, Hongmei Zhu, Liqiong Zhang, Yihong Fu, Jiamin Liu,
Zhenchao Wang & Guiping OuYang
To cite this article: Junhu Qin, Mei Zhu, Hongmei Zhu, Liqiong Zhang, Yihong Fu, Jiamin Liu,
Zhenchao Wang & Guiping OuYang (2020): Synthesis and antitumor activity of novel pyridazinone
derivatives containing 1,3,4-thiadiazole moiety, Phosphorus, Sulfur, and Silicon and the Related
Elements, DOI: 10.1080/10426507.2020.1737062
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2020.1737062
Synthesis and antitumor activity of novel pyridazinone derivatives containing
1,3,4-thiadiazole moiety
Junhu Qina,b†, Mei Zhub†, Hongmei Zhub, Liqiong Zhangb, Yihong Fub, Jiamin Liub, Zhenchao Wangb,c , and
Guiping OuYangb,c
b
aGuizhou Institute of Environmental Sciences Research and Design, Guiyang, China; College of Pharmacy, Guizhou University, Guiyang,
c
China; Guizhou Engineering Laboratory for Synthetic Drugs, Guizhou University, Guiyang, China
ABSTRACT
ARTICLE HISTORY
Received 14 November 2019
Accepted 27 February 2020
A series of novel pyridazinone derivatives containing the 1,3,4-thiadiazole moiety were synthesized
and characterized by H NMR, 13C NMR, spectroscopies HRMS and IR. Among them, the structure
1
of compound 5c (2-(Tert-butyl)ꢀ4-chloro-5-((5-((2-ethylphenyl)amino)ꢀ1,3,4-thiadiazol-2-yl)thio)pyr-
idazin-3(2H)-One) was unambiguously confirmed via single crystal X-ray diffraction analysis.
The inhibitory activity of all the target compounds against MGC-803 and Bcap-37 was determined
by MTT assay, with doxorubicin (the inhibition rates were 95.5 0.4% and 95.7 1.0% respectively)
as a control. The preliminary results showed that the inhibitory activity of compound 5n
(2-(Tert-butyl)ꢀ4-chloro-5-((5-((3-fluorophenyl)amino)ꢀ1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-One)
was superior to the others. The inhibition rates of MGC-803 and Bcap-37 cells were 86.3 2.2%
and 92.3 0.6% at a concentration of 10 lmol/L, respectively. The preliminary structure-activity
relationship showed that when the 2-position of the benzene ring was substituted by a methyl
group, such as compound 5j (2-(Tert-butyl)ꢀ4-chloro-5-((5-((2,3-dimethylphenyl)amino)ꢀ1,3,4-
thiadiazol-2-yl)thio)pyridazin-3(2H)-One), it exhibited good anticancer activity on MGC-803 cells.
Besides, introducing fluorine, chlorine, or trifluoromethyl group onto the benzene ring, such as
compound 5 m (2-(Tert-butyl)ꢀ4-chloro-5-((5-((4-(trifluoromethoxy)phenyl)amino)ꢀ1,3,4-thiadiazol-
2-yl)thio)pyridazin-3(2H)-One), displayed good anticancer activity on MGC-803 and Bcap-37 cells.
KEYWORDS
Pyridazinone derivatives;
1,3,4-thiadiazole; antitumor
activity; structure-activity
relationship
GRAPHICAL ABSTRACT
Introduction
cardiotonic for the treatment of cardiac insufficiency and
heart failure.[19,20] This six membered ring compound has
many different physiological functions and can be used in
the treatment of different diseases. At the same time, studies
showed that derivatives containing 1,3,4-thiadiazole groups
have good biological properties, such as antibacterial,[21–23]
anticancer[24–29] and other activities,[30] among them, the
biological activity of 1,3,4-thiadiazole sulfide compounds is
particularly prominent.[31,32] This may be due to the fact
that the thioether bond reduces the lipophilicity of the com-
pound and improves the biological activity of the compound
as a hydrogen bond receptor.
The chemical properties of pyridazinones have been an
interesting area of research for many years. Pyridazinone
derivatives are an important class of nitrogen-containing
six-membered heterocyclic compounds and are promising
lead compounds for the research and development of new
pesticides and pharmaceuticals.[1–5] These derivatives are
widely used in pesticide research, as pesticides, fungicides,
herbicides, and plant growth regulators, and are attracting
additional attention due to their broad spectrum of bio-
logical activity and structural diversity.[6–11] In the field of
medicine, many derivatives containing a pyridazinone struc-
ture have anti-inflammatory,[12–14] anti-platelet aggrega-
tion,[15,16] and anti-cancer[17,18] activities. Some of these
According to a group of data released by the World
Health Organization, cancer incidence is still high all over
compounds can even be used as calcium sensitizers and the world. At present, chemotherapy is the main means to
CONTACT Zhenchao Wang
wzc.4884@163.com
College of Pharmacy, Guizhou University, Guiyang, P. R. China.
†These authors contributed equally to this work.
Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/10426507.2020.1737062.
ß 2020 Taylor & Francis Group, LLC

2
J. QIN ET AL.
Scheme 1. Synthetic route of compounds 5a – 5n.
solve the world problem of cancer, but the existing drugs
will bring some side effects and drug resistance.[33,34]
Therefore, a search for low toxicity and high-efficiency che-
motherapeutic drugs has been a major topic in the field of
medical research. Based on the above description, we want
to splice the two active groups together to explore their anti-
tumor activity. Thus, a series of novel pyridazinone deriva-
tives containing 1,3,4-thiadiazole sulfide groups were
designed and synthesized (Scheme 1). The structures of the
The structures of all the target compounds were charac-
terized by their spectral data. In the IR spectrum of com-
pounds 5a ꢀ 5n, the characteristic absorption peaks near
3450 ꢀ 3200 cmꢀ1
,
2980 ꢀ 2830 cmꢀ1
,
1700 ꢀ 1650 cmꢀ1
,
1600 ꢀ 1450 cmꢀ1, confirmed the presence of N-H, Tert-
butyl C-H, C ¼ O functional groups and Ar-H, respectively.
1
In the H NMR spectrum of the target compounds, the N-H
proton appeared as a sharp singlet at 11.00 ꢀ 9.00 ppm; the
proton of pyridazinone ring appeared as a sharp singlet at
near 7.50 ppm; the aromatic protons revealed the expected
pattern near 8.00 ꢀ 6.80 ppm, the three-methyl protons sig-
nal of Tert-butyl was observed as a singlet around 1.56 ppm.
In the 13C NMR spectrum of the target compounds, the
carbon atom on a thiadiazole ring attached to an out-of-ring
sulfur atom appeared at 169 ꢀ 174 ppm, the carbon atom of
C ¼ O on pyridazinone ring was observed around
155.4 ppm, the carbon atom on a thiadiazole ring closed to
amino appeared at 140.8 ꢀ 144.6 ppm, the carbon atoms on
benzene ring were observed at 105 ꢀ 140 ppm, the Tertiary
carbon atom was observed around 66.4 ppm, the primary
carbon atom on Tert-butyl was observed around 27.8 ppm,
and the carbon atoms of substituted group on the benzene
ring were observed at 15.0 ꢀ 24.3 ppm. Taking compound 5a
for example, in the IR spectrum, the characteristicꢀa1bsorp-
1
target compounds were confirmed by H NMR, 13C NMR,
HRMS and IR spectroscopies.
Results and discussion
Chemistry
The synthetic route to the target compounds is demon-
strated in Scheme 1. The intermediate of 2-(Tert-butyl)ꢀ4,5-
dichloropyridazin-3(2H)-one (1) was prepared by reacting
mucochloric acid with Tert-butylhydrazine.[35] The aryliso-
thiocyanate (2) was synthesized by a nucleophilic substitu-
tion reaction of dithiocarbamate salt, which was previously
prepared from substituted aniline reacting with carbon
disulfide in triethylamine at ice bath condition for 12 h, with
ethyl chloroformate (dissolving in chloroform) in the pres-
ence of triethylamine at room temperature stirred for 6 h.
Then, to the resulting isothiocyanate, 80% hydrazine hydrate
dissolved in isopropanol was dropwise added and stirred for
2 h at room temperature to afford N-(substituted phenyl)hy-
drazinecarbothioamide (3).[36] The 5-((substituted phenyl)-
amino)ꢀ1,3,4-thiadiazole-2-thiol (4) was prepared by
reaction of N-(substituted phenyl)hydrazinecarbothioamide,
carbon disulfide and potassium hydroxide in anhydrous
ethanol for refluxing 4 h.[37] Finally, the target compounds
5a ꢀ 5n were obtained by reacting 5-((substituted phenyl)-
tion peaks at 3439 cmꢀ1
,
3064 cmꢀ1
,
1602 cmꢀ1 and 1587 cmꢀ1 and
,
2961 cm
and
2930 cmꢀ1 1660 cmꢀ1
,
1456 cmꢀ1, confirmed the presence of N-H, Tert-butyl C-H,
two methyl groups on benzene ring C-H, C ¼ O functional
groups and three hydrogen on benzene ring. In the 1H
NMR spectrum of the compound 5a, the N-H proton
appeared as a sharp singlet at 10.70 ppm, the proton of pyri-
dazinone ring appeared as a sharp singlet at 7.49 ppm; the
aromatic protons at 7.22 ppm and 6.72 ppm. In the 13C
NMR spectrum of the compound 5a, the carbon atom on a
thiadiazole ring attached to an out-of-ring sulfur atom
appeared at 169.6 ppm, the another carbon atom on a thia-
diazole ring was observed at 142.5 ppm, the carbon atom of
C ¼ O was observed at 155.4 ppm, the carbon atom at the 5-
position of pyridazin-3(2H)-one was observed at 140.3 ppm,
the carbon on benzene ring attached to N-H was observed
amino)ꢀ1,3,4-thiadiazole-2-thiol
(4)
with
2-(Tert-
butyl)ꢀ4,5-dichloropyridazin-3(2H)-one (1) in acetone in
the presence of anhydrous K2CO3 at room temperature for
6 ꢀ 8 h in good yields.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
Figure 1. Drawing of 2-(Tert-butyl)-4-chloro-5-((5-((2-ethylphenyl)amino)-1,3,4 -thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5c).
C36H40Cl2N10O2S4 (M ¼ 843.92 g/mol) was as follows
(Table 1): triclinic, space group P-1 (no. 2), a ¼ 10.5705(7)
Å, b ¼ 13.3340(9) Å, c ¼ 18.2385(12) Å, a ¼ 100.945(2),
b ¼ 104.517(2), c ¼ 99.652(2), V ¼ 2379.8(3) Å3, Z ¼ 2,
T ¼ 293(2) K, l (MoKa) ¼ 0.352 mmꢀ1, Dcalc ¼ 1.178 g/
cm3, 24385 reflections measured (4.084 ꢁ 2⍜ ꢁ 49.998) ,
8329 unique (Rint ¼ 0.0331, Rsigma ¼ 0.0360) which were
used in all calculations. The final R1 was 0.0579 (I > 2r(I))
and wR2 was 0.1996. Further details of the bond lengths,
bond angles were are shown in Table S 1 and Table S 2
(Supplemental Materials)
Table 1. Crystal structure and data refinement parameters.
Compound
5c
Empirical Formula
Formula Weight
Crystal System / Space Group
C36H40Cl2N10O2S4
843.92
Triclinic / P-1
10.5705(7)
13.3340(9)
18.2385(12)
100.945(2)
104.517(2)
99.652(2)
2379.8(3)
2
a / Å
b / Å
c / Å
ꢂ
a /
ꢂ
b / ꢂ
V / Å3
Z
c /
D
(g/cm3)
1.178
0.352
calc
l (mmꢀ1
)
Crystal size (mm)
Color / Shape
Temp (K)
0.23 ꢃ 0.22 ꢃ 0.21
Light yellow / block
293(2)
Antitumor activity assay
The cytotoxic activity was evaluated for all of the target
compounds 5a ꢀ 5n against the MGC-803(Human gastric
carcinoma cell line) and Bcap-37(Human breast cancer cell
line) cell lines by MTT assay in vitro. Adriamycin (ADM)
and the compounds were dissolved in DMSO. The control
cell was treated with culture medium containing 0.1%
DMSO. ADM was used as a positive control. The inhibitory
percentage of cells treated with 10 mmol/L and 1 mmol/L of
each compound for 72 h. The results are summarized in
Table S 3. As shown, most of the target compounds dis-
played good inhibitory bioactivity against the MGC-803 and
Bcap-37 cancer cells in vitro. At a concentration of 10 lmol/
L, among these compounds, 5c, 5j, 5 m, 5n showed inhibi-
tory ratios against MGC-806 were 86.7%, 86.4%, 86.9%,
86.3%, and compound 5k and 5n against Bcap-37 showed
83.6%, 92.3% inhibitory ratios, respectively. Preliminary
structure-activity relationship showed that when the 2-pos-
Theta range for collection
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness of fit on F2
Final R indices [I > 2r(I)]
R indices (all data)
2.2108 ꢁ h ꢁ 21.6944
24385
5765
533
1.067
0.0579
0.1996
Largest difference peak/hole
0.556 / ꢀ0.407
at 139.1 ppm, the two carbon atoms at the methyl substitu-
tion on the benzene ring were observed at 138.9 ppm, the
carbon atoms at the 6-position and the 4-position of pyrida-
zin-3(2H)-one were observed at 131.2 ppm and 130.6 ppm,
respectively. The carbon atom at the 4-position of benzene
ring was observed at 125.1 ppm, and the 2-position and 6-
position carbon atoms on the benzene ring were observed at
116.3 ppm, the Tertiary carbon atom was observed at
66.4 ppm, the primary carbon atom on Tert-butyl was
observed at 27.8 ppm, and the two methyl carbons on the ition of the benzene ring was substituted by a methyl group,
benzene ring were observed at 21.7 ppm.
5j exhibited good anticancer activity toward MGC-803 cells;
A single crystal X-ray analysis revealed the structure of when the fluorine, chlorine, or trifluoromethyl groups were
the product 5c as shown in Figure 1. The crystal data of introduced into benzene ring, such as compound 5n, good

4
J. QIN ET AL.
inhibitory bioactivity toward MGC-803 and Bcap-37 cancer 8 h, and the reaction was monitored by TLC. After comple-
cells was exhibited.
tion of the reaction, the solvent was removed under reduced
pressure, and distilled water (10 mL) was added to the mix-
ture to remove potassium carbonate. Residues were purified
by silica gel column chromatography (EtOAc/petroleum
ether increased from 1/4 to 1/2). The physical and spectral
data of 5a ꢀ 5n are as follows.
Experimental
General methods
Unless otherwise stated, all the reagents and reactants were
purchased from commercial suppliers; melting points were
determined using an XT-4 binocular microscope (Beijing
Tech Instrument, China) and was not corrected. The 1H
NMR and 13C NMR spectra were recorded on a JEOL ECX
500 NMR spectrometer at room temperature operating at
Characterization of compounds 5a 2 5n
2-(Tert-butyl)-4-chloro-5-((5-((3,5-dimethylphenyl)amino)-
1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5a)
White solid; Yield: 42.0%; m.p. 228 ꢀ 230 ꢂC; IR (KBr) ꢀ
1
500 MHz for H NMR and 125 MHz for 13C NMR, using
(cmꢀ1): 3439, 3065, 2961, 2930, 1660, 1602, 1587, 1456,
DMSO-d6 as the solvent and TMS as an internal standard;
infrared spectra were recorded with KBr on a Bruker
VECTOR 22 spectrometer; elemental analysis was performed
on an Elemental Vario-III CHN analyzer; MS analysis was
performed on Agilent LC-MSD Trap VL Apparatus. The
course of the reactions was monitored by TLC; analytical
TLC was performed on silica gel GF254; column chromato-
graphic purification was carried out using silica gel GF254
under normal pressure. The Supplemental Materials contain
sample 1H NMR, 13C NMR, HRMS spectra for 5a ꢀ 5n
(Figures S 1 – S 41) and optimization results of compound
5d synthesis (Table S 4 and Table S 5).
1
1366, 1267, 1209, 1103, 935, 847, 785; H NMR (500 MHz,
DMSO-d6) d: 10.70 (s, 1H, NH), 7.49 (s, 1H, NCH), 7.22 (s,
2H, Ph-H), 6.72 (s, 1H, Ph-H), 2.28 (s, 6H, 2CH3), 1.56 (s,
9H, 3CH3); 13C NMR (125 MHz, DMSO-d6) d: 169.6, 155.4,
142.5, 140.3, 139.1, 138.9, 131.2, 130.6, 125.1, 116.3, 66.4,
27.8, 21.7; HRMS (ESI) found: 444.1 (M＋Na)þ; Anal.Calcd
for C18H20ClN5OS2 (g): C, 51.23; H, 4.78; N, 16.60; Found
C, 51.14; H, 4.80; N, 16.02.
2-(Tert-butyl)-4-chloro-5-((5-(p-tolylamino)-1,3,4-thiadia-
zol-2-yl)thio)pyridazin-3(2H)-one (5 b)
White crystal; Yield: 38.2%; m.p. 219 ꢀ 221 ꢂC; IR (KBr) ꢀ
(cmꢀ1): 3260, 3070, 2974, 1667, 1607, 1549, 1520, 1449,
1391, 1263, 1142, 1096, 936, 812; 1H NMR (500 MHz,
General procedure for the preparation of intermediate 1
A
solution of tert-butylhydrazine hydrochloride 3.74 g DMSO-d6) d: 10.73 (s, 1H, NH), 7.51 (s, 1H, NCH),
(30 mmol) and sodium acetate (2.46 g, 30 mmol) in water 7.49 ꢀ 7.46 (br, m, 2H, Ph-H), 7.19 (d, J ¼ 8.1 Hz, 2H, Ph-
(60 mL) was stirred at 10 ꢂC. Mucochloric acid (4.22 g, H), 2.28 (s, 3H, CH3), 1.56 (s, 9H, 3CH3); 13C NMR
25 mmol)) was added slowly. A yellow precipitate formed after
stirring for 1 h at 10 ꢂC. Then toluene (50 mL) was added and
stirring was continued for a further 6 h at 40 ꢂC. After comple-
tion of the reaction, the organic phase was washed with 15%
NaOH solution (5 mL) and brine (10 mL), and dried with
anhydrous Na2SO4. Removal of the solvent under reduced
pressure, gave a crude product. The residue was recrystallized
from ethanol to obtain the intermediate 2-(Tert-butyl)ꢀ4,5-
dichloropyridazine-3(2H)-one (1), which as a yellowish crys-
tal. m.p. 64 ꢀ 66 ꢂC (lit.[35], m.p. 64 ꢂC), yield 62.7%.
(125 MHz, DMSO-d6) d: 169.8, 155.4, 142.3, 139.1, 138.0,
132.5, 131.1, 130.5, 130.2, 118.7, 66.4, 27.8, 20.9; HRMS
(ESI) found: 430.1 (M＋ Na)þ; Anal.Calcd for
C17H18ClN5OS2 (g): C, 50.05; H, 4.45; N, 17.17; Found C,
50.00; H, 4.39; N, 17.24.
2-(Tert-butyl)-4-chloro-5-((5-((2-ethylphenyl)amino)-1,3,4-
thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5c)
Light yellow solid; Yield: 40.6%; m.p. 138 ꢀ 140 ꢂC; IR (KBr)
ꢀ (cmꢀ1): 3294, 3055, 2965, 2874, 1649, 1589, 1541, 1489,
1
1452, 1395, 1366, 1211, 1099, 935, 752; H NMR (500 MHz,
General procedure for the preparation intermediate 4
DMSO-d6) d: 10.05 (s, 1H, NH), 7.80 (d, J ¼ 8.0 Hz, 1H, Ph-
H), 7.49 (s, 1H, NCH), 7.30 (d, J ¼ 7.5 Hz, 1H, Ph-H), 7.26
(t, J ¼ 7.7 Hz, 1H, Ph-H), 7.17 (t, J ¼ 7.5 Hz, 1H, Ph-H),
2.70 ꢀ 2.65 (q, J ¼ 7.5, 2H, CH2), 1.56 (s, 9H, 3CH3), 1.54 (t,
J ¼ 7.5 Hz, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) d:
172.8, 155.3, 141.8, 139.3, 138.4, 137.0, 131.0, 130.3, 129.7,
127.4, 126.2, 123.8, 66.4, 27.8, 24.3 15.0; HRMS (ESI) found:
444.1 (M＋ Na)þ; Anal.Calcd for C18H20ClN5OS2 (g): C,
51.23; H, 4.78; N, 16.60; Found C, 51.28; H, 4.57; N, 16.67.
The intermediate 4 (5-((substituted phenyl)amino)ꢀ1,3,4-
thiadiazole-2-thio) were prepared according to the literature
procedure[352–37] from substituted aniline as the starting
material through salt formation, substitution, hydrazinolysis,
cyclization, and acidification. All compounds were synthesized
under mild conditions with moderate yields.
General procedure for the preparation of 5a 2 5n
The intermediate 1 (1 mmol) and anhydrous potassium car- 2-(Tert-butyl)-4-chloro-5-((5-((2,5-dimethylphenyl)amino)-
bonate (1.5 mmol) were added to acetone solution (25 mL) 1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5d)
and stirred. Intermediate 4 (1 mmol) was added to the reac- White solid; Yield: 42.0%; m.p. 220 ꢀ 222 ꢂC; IR (KBr) ꢀ
tion solution in batches are 30 min at room temperature for (cmꢀ1): 3182, 3021, 2963, 2922, 1653, 1557, 1501, 1456,

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
5
1427, 1265, 1140, 934, 822, 785; 1H NMR (500 MHz, 1450, 1368, 1265, 1126, 867, 752; 1H NMR (500 MHz,
DMSO-d6) d: 9.97 (s, 1H, NH), 7.65 (s, 1H, Ph-H), 7.48 (s, DMSO-d6) d: 10.83 (s, 1H, NH), 7.51 (s, 1H, NCH), 7.31 (s,
1H, NCH), 7.14 (d, J ¼ 7.4 Hz, 1H, Ph-H), 6.92 (d, 1H, Ph-H), 7.28 (d, J ¼ 8.1 Hz, 1H, Ph-H), 7.11 (d,
J ¼ 7.4 Hz, 1H, Ph-H), 2.28 (s, 3H, CH3), 2.24 (s, 3H, CH3), J ¼ 7.5 Hz, 1H, Ph-H), 6.67-6.65 (m, 1H, Ph-H), 3.77 (s, 3H,
1.56 (s, 9H, 3CH3); 13C NMR (125 MHz, DMSO-d6) d: OCH3), 1.56 (s, 9H, 3CH3); 13C NMR (125 MHz, DMSO-
172.1, 155.4, 142.0, 139.2, 138.8, 136.5, 131.2, 131.1, 130.4, d6) d: 169.5, 160.5, 155.4, 143.0, 141.5, 139.0, 131.2, 130.6,
127.4, 126.2, 123.1, 66.4, 27.8, 21.3, 18.0; HRMS (ESI) found: 128.6, 110.9, 108.7, 104.5, 66.4, 55.6, 27.8; HRMS (ESI)
444.1 (M＋ Na)þ; Anal.Calcd for C18H20ClN5OS2 (g): C, found: 446.1 (M＋ Na)þ; Anal.Calcd for C17H18ClN5O2S2
51.23; H, 4.78; N, 16.60; Found C, 51.47; H, 4.71; N, 16.72.
(g): C, 48.16; H, 4.28; N, 16.52; Found C, 48.25; H, 4.06;
N, 16.76.
2-(Tert-butyl)-4-chloro-5-((5-((2,4-dimethylphenyl)amino)-
1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5e)
2-(Tert-butyl)-4-chloro-5-((5-((2,6-dimethylphenyl)amino)-
White solid; Yield: 38.9%; m.p. 170 ꢀ 172 ꢂC; IR (KBr) ꢀ 1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5i)
(cmꢀ1): 3169, 3030, 2982, 2926, 1653, 1589, 1553, 1504, White solid; Yield: 41.6%; m.p. 208 ꢀ 210 ꢂC; IR (KBr) ꢀ
1456, 1425, 1387, 1209, 1099, 934, 845, 785; 1H NMR (cmꢀ1): 3163, 3019, 2974, 2922, 1667, 1597, 1541, 1506,
1
(500 MHz, DMSO-d6) d: 9.99 (s, 1H, NH), 7.64 (d, 1488, 1422, 1388, 1265, 1206, 1142, 935, 779, 750; H NMR
J ¼ 8.6 Hz, 1H, Ph-H), 7.47 (s, 1H, NCH), 7.09 (s, 1H, Ph- (500 MHz, DMSO-d6) d: 10.02 (s, 1H, NH), 7.41 (s, 1H,
H), 7.05(d, J ¼ 8.6 Hz, 1H, Ph-H), 2.27 (s, 3H, CH3), 2.24 (s, NCH), 7.17 (s, 3H, Ph-H), 2.23 (s, 6H, 2CH3), 1.56 (s, 9H,
3H, CH3), 1.56 (s, 9H, 3CH3); 13C NMR (125 MHz, DMSO- 3CH3); 13C NMR (125 MHz, DMSO-d6) d: 173.9, 155.3,
d6) d: 172.7, 155.4, 141.5, 139.3, 136.6, 135.1, 132.0, 131.0, 140.8, 139.3, 137.8, 135.9, 130.8, 130.1, 129.1, 128.1, 66.4,
130.3, 127.8, 126.1, 123.2, 66.4, 27.8, 21.0, 18.3; HRMS (ESI) 27.8, 18.4; HRMS (ESI) found: 444.1 (M＋Na)þ; Anal.Calcd
found: 444.1 (M＋Na)þ; Anal.Calcd for C18H20ClN5OS2 (g): for C18H20ClN5OS2 (g): C, 51.23; H, 4.78; N, 16.60; Found
C, 51.23; H, 4.78; N, 16.60; Found C, 51.12; H, 4.86; C, 51.57; H, 4.28; N, 16.28.
N, 16.72.
2-(Tert-butyl)-4-chloro-5-((5-((2,3-dimethylphenyl)amino)-
2-(Tert-butyl)-4-chloro-5-((5-((3-chlorophenyl)amino)-1,3,4-
thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5f)
1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5j)
White solid; Yield: 40.0%; m.p. 228 ꢀ 230 ꢂC; IR (KBr) ꢀ
Light yellow solid; Yield: 39.0%; m.p. 222 ꢀ 224 ꢂC; IR (KBr) ꢀ (cmꢀ1): 3183, 3019, 2963, 2868, 1653, 1557, 1526, 1456, 1427,
(cmꢀ1): 3277, 3088, 2980, 2932, 1635, 1597, 1554, 1489, 1450, 1368, 1265, 1209, 1096, 932, 822, 783; H NMR (500 MHz,
1
1
1364, 1207, 1146, 935, 771, 690; H NMR (500 MHz, DMSO- DMSO-d6) d: 9.98 (s, 1H, NH), 7.48 (s, 1H, NCH), 7.37 (m,
d6) d: 10.96 (s, 1H, NH), 7.84 (s, 1H, Ph-H), 7.47 (s, 1H, 1H, Ph-H), 7.14 (d, J ¼ 8.0 Hz, 1H, Ph-H), 6.92 (d, J ¼ 7.5 Hz,
NCH), 7.39 ꢀ 7.35(m, 2H, Ph-H), 7.06 (d, J ¼ 8.0 Hz, 1H, Ph- 1H, Ph-H), 2.28 (s, 3H, CH3), 2.24 (s, 3H, CH3), 1.56 (s, 9H,
H), 1.51 (s, 9H, 3CH3); 13C NMR (125 MHz, DMSO-d6) d: 3CH3); 13C NMR (125 MHz, DMSO-d6) d: 172.1, 155.4, 141.9,
169.0, 155.4, 144.2, 141.6, 138.9, 134.1, 131.4, 131.3, 130.9, 139.3, 138.8, 136.5, 131.2, 131.0, 130.4, 127.3, 126.2, 123.1,
122.9, 117.9, 116.9, 66.5, 27.7; HRMS (ESI) found: 450.0 (M＋ 66.4, 27.8, 21.3, 18.0; HRMS (ESI) found: 444.1 (M＋ Na)þ;
Na)þ; Anal.Calcd for C16H15Cl2N5OS2 (g): C, 44.86; H, 3.53; Anal.Calcd for C18H20ClN5OS2 (g): C, 51.23; H, 4.78; N, 16.60;
N, 16.35; Found C, 44.84; H, 3.50; N, 16.39.
Found C, 51.14; H, 4.40; N, 16.67.
2-(Tert-butyl)-4-chloro-5-((5-((2-chlorophenyl)amino)-1,3,4-
thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5 g)
2-(Tert-butyl)-4-chloro-5-((5-((2,5-dimethoxyphenyl)amino)-
1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5k)
White solid; Yield: 38.8%; m.p. 230 ꢀ 232 ꢂC; IR (KBr) ꢀ Light yellow solid; Yield: 40.2%; m.p. 138 ꢀ 140 ꢂC; IR (KBr)
(cmꢀ1): 3445, 3111, 2987, 2922, 1651, 1582, 1547, 1474, ꢀ (cmꢀ1): 3242, 3073, 2963, 2828, 1643, 1601, 1541, 1474,
1450, 1368, 1267, 1103, 935, 758; 1H NMR (500 MHz, 1451, 1368, 1223, 1045, 941, 787; 1H NMR (500 MHz,
DMSO-d6) d: 10.31 (s, 1H, NH), 8.29 (d, J ¼ 8.0 Hz, 1H, Ph- DMSO-d6) d: 10.26 (s, 1H, NH), 8.05 (s, 1H, Ph-H), 7.49 (s,
H), 7.54 (d, J ¼ 8.0 Hz, 1H, Ph-H), 7.52(s, 1H, NCH), 7.42- 1H, NCH), 7.00 (d, J ¼ 8.6 Hz, 1H, Ph-H), 6.64 ꢀ 6.61 (m,
7.39 (m, 1H, Ph-H), 7.18-7.15 (m, 1H, Ph-H), 1.57 (s, 9H, 1H, Ph-H), 3.84 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 1.56 (s,
3CH3); 13C NMR (125 MHz, DMSO-d6) d: 170.1, 155.4, 9H, 3CH3); 13C NMR (125 MHz, DMSO-d6) d: 169.4, 155.4,
144.6, 139.0, 137.0, 131.2, 130.7, 130.3, 128.5, 125.6, 124.0, 153.8, 144.0, 143.1, 139.1, 131.2, 130.7, 130.1, 112.5, 107.5,
123.1, 66.4, 27.8; HRMS (ESI) found: 450.0 (M＋ Na)þ; 107.0, 66.4, 56.9, 56.0, 27.8; HRMS (ESI) found: 476.1 (M＋
Anal.Calcd for C16H15Cl2N5OS2 (g): C, 44.86; H, 3.53; N, Na)þ; Anal.Calcd for C18H20ClN5O3S2 (g): C, 47.62; H, 4.44;
16.35; Found C, 45.16; H, 3.57; N, 16.76.
N, 15.43; Found C, 47.45; H, 4.50; N, 15.47.
2-(Tert-butyl)-4-chloro-5-((5-((3-methoxyphenyl)amino)-
1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5 h)
(R)-2-(tert-butyl)-4-chloro-5-((5-((1-phenylethyl)amino)-
1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5 l)
White solid; Yield: 38.0%; m.p. 194 ꢀ 196 ꢂC; IR (KBr) ꢀ Pink solid; Yield: 37.8%; m.p. 161 ꢀ 163 ꢂC; IR (KBr) ꢀ (cmꢀ1):
(cmꢀ1): 3240, 3049, 2960, 2878, 1651, 1614, 1568, 1497, 3179, 3065, 2967, 2878, 1659, 1568, 1503, 1454, 1366, 1263,

6
J. QIN ET AL.
1
1209, 1138, 933, 783, 702; H NMR (500 MHz, DMSO-d6) d: Antitumor activity assay
8.96 (d, J ¼ 6.9 Hz, 1H, NH), 7.40 (s, 1H, NCH), 7.39ꢀ 7.27 (br,
Cell lines and culture
Gastric cancer cell line (MGC-803) and breast cancer cell
m, 5H, Ph-H), 4.91 ꢀ 4.85 (m, 1H, CH), 1.55 (s, 9H, 3CH3),
1.49 (d, J ¼ 6.9 Hz, 1H, CH3); 13C NMR (125 MHz, DMSO-d6)
d: 172.9, 155.3, 143.9, 140.0, 139.4, 130.7, 130.0, 129.1, 127.8,
126.6, 66.4, 55.2, 27.8, 23.6; HRMS (ESI) found: 422.1 (M＋
H)þ; Anal.Calcd for C18H20ClN5OS2 (g): C, 51.23; H, 4.78; N,
16.60; Found C, 51.14; H, 4.70; N, 16.67.
line (Bcap-37) were obtained from the Institute of
Biochemistry and Cell Biology, China Academy of Science.
All cell lines were cultured in PRMI 1640 medium and sup-
plemented with 10% heat-inactivated fetal bovine serum
(FBS) in a humidified atmosphere of 5% CO2 at 37 ꢂC.
MTT assay
2-(Tert-butyl)-4-chloro-5-((5-((4-(trifluoromethoxy)phenyl)a-
mino)-1,3,4-thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5 m)
Pink solid; Yield: 37.1%; m.p. 190 ꢀ 192 ꢂC; IR (KBr) ꢀ
(cmꢀ1): 3279, 3092, 2986, 2887, 1638, 1609, 1566, 1508,
Antiproliferative effects were identification with tetrazolium
dye assay (MTT). All tested compounds were dissolved in
DMSO (1-100 lM solution) and subsequently diluted in the
culture medium before treatment of the cultured cells. Tested
cells were plated in 96-well plates at a density of 2 ꢃ 103 cells/
well/100 lL of the proper culture medium and treated with the
compounds at 1 and 10lM for 72h. In parallel, the cells treated
with 0.1% DMSO served as control. Finally, 100 lL of MTT was
added, and the cells were incubated for 4 h. The MTT-formazan
formed by metabolically viable cells was dissolved in 100 lL of
SDS for 12h. The absorbance was then measured at 595 nm
with a micro plate reader (BIO-RAD, model 680), which is dir-
ectly proportional to the number of living cells in culture. The
percentage of cytotoxicity was calculated using the formula.
Tissue culture reagents were obtained from Gibco BRL. The
experiment was performed in triplicate. The percentage of cyto-
toxicity was calculated using this formula.
1
1508, 1454, 1368, 1204, 1144, 935, 843; H NMR (500 MHz,
DMSO-d6) d: 11.00 (s, 1H, NH), 7.75 (d, J ¼ 8.6 Hz, 2H, Ph-
H), 7.52 (s, 1H, NCH), 7.40(d, J ¼ 8.6 Hz, 2H, Ph-H), 1.56
(s, 9H, 3CH3); 13C NMR (125 MHz, DMSO-d6) d: 169.2,
155.4, 143.8, 143.6, 139.6, 139.0, 131.2, 130.7, 122.7,119.8,
119.6, 66.4, 27.8; HRMS (ESI) found: 478.0 (M＋ H)þ;
Anal.Calcd for C17H15ClF3N5O2S2 (g): C, 42.72; H, 3.16; N,
14.65; Found C, 42.34; H, 3.20; N, 14.37.
2-(Tert-butyl)-4-chloro-5-((5-((3-fluorophenyl)amino)-1,3,4-
thiadiazol-2-yl)thio)pyridazin-3(2H)-one (5n)
Light yellow solid; Yield: 40.8%; m.p. 204 ꢀ 206 ꢂC; IR (KBr)
ꢀ (cmꢀ1): 3287, 3094, 2988, 1648, 1609, 1566, 1493, 1452,
1368, 1261, 1140, 961, 847, 785; 1H NMR (500 MHz,
DMSO-d6) d: 11.02 (s, 1H, NH), 7.67 (d, J ¼ 11.5 Hz, 1H,
Ph-H), 7.53 (s, 1H, NCH), 7.43-7.41 (m, 2H, Ph-H), 7.32 (d,
J ¼ 8.0 Hz, 1H, Ph-H), 6.91 ꢀ 6.88 (m, 1H, Ph-H), 1.56 (s,
9H, 3CH3); 13C NMR (125 MHz, DMSO-d6) d: 169.0, 164.0,
155.4, 144.0, 138.9, 131.4, 130.8, 128.3, 121.5, 114.3, 109.5,
105.3, 66.4, 27.8; HRMS (ESI) found: 434.1 (M＋ Na)þ;
Anal.Calcd for C16H15ClFN5OS2 (g): C, 46.65; H, 3.67; N,
17.00; Found C, 46.65; H, 3.70; N, 17.01.
ðControlabs ꢀ BlankabsÞ ꢀ ðTestabs ꢀ BlankabsÞ
%Cytotoxicity ¼
ꢃ 100
ðControlabs ꢀ BlankabsÞ
Conclusion
A series of novel pyridazinone derivatives containing a 1,3,4-
thiadiazole moiety were synthesized, characterized, and their
preliminary biological activities were studied. The target
compounds were synthesized starting from mucochloric acid,
substituted hydrazine, and aromatic amines. The structures
of all compounds were 1H NMR, 13C NMR, IR and MS
spectroscopies confirmed, at the same time, the synthesis
conditions of the target compound were optimized, and the
optimal synthesis conditions were determined: under the
condition of room temperature, acetone as solvent, anhyd-
rous potassium carbonate as acid-binding agent, reaction 6-
8 h, a better yield can be obtained. Further, the compound 5c
was subjected to single-crystal culture and crystal structure
characterization. Using doxorubicin as control, MTT assay
was used to determine the inhibitory effect of the compound
on the proliferation of human gastric cancer cells (MGC-
803) and human breast cancer cells (BCAP-37) in vitro. The
results showed that the concentration was 10 lmol/L, most
of the target compounds showed good anticancer activity.
However, the number of synthetic target combinations is
limited, and the structure is relatively simple; further work
Crystal structure determination and refinement
A single crystal was grown for compound 5c from mixed
MeOH/CH2Cl2 solution by slow evaporation. Crystallographic
data for 5c were collected on a Bruker Smart APEX II CCD
diffractometer with graphite-monochromated Mo Ka radi-
ation (k ¼ 0.71073 Å) in the x scan mode. The crystal was kept
at 293(2) K during data collection. Using Olex2,[38] the struc-
ture was solved with the ShelXD[39] structure solution pro-
gram using Dual Space and refined with the ShelXL[40]
refinement package using Least Squares minimization. Data
for the structures reported here have been deposited with the
Cambridge Crystallographic Data Center with deposition
numbers CCDC1984502 which contain the supplementary
crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic will optimize the structure of better-active compounds and is
Data Center via www.ccdc.cam.ac.uk/data_request/cif. expected to screen for a lead compound with high activity.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
7
Pyridazinone. Eur. J. Med. Chem 2016, 123, 256–281. DOI: 10.
1016/j.ejmech.2016.07.061.
Funding
This research was financially supported by the project of State Key
Laboratory of Functions and Applications of Medicinal Plants,
Guizhou Medical University, (No. FAMP201707K), Guizhou Science
Technology Program (No. 20181051), and the National Natural Science
Foundation of China (No. 21867004).
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ORCID
Zhenchao Wang
http://orcid.org/0000-0003-1859-0128
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