Design, synthesis, biological evaluation and inhibition mechanism of 3-/4-alkoxy phenylethylidenethiosemicarbazides as new, potent and safe tyrosinase inhibitors

Article abstract of DOI:10.1248/cpb.c19-00949

Tyrosinase plays important roles in many different disease related processes, and the development of its inhibitors is particularly important in biotechnology. In this study, thirty-nine 3-/4-alkoxyphenylethyli-denethiosemicarbazides were synthesized as novel tyrosinase inhibitors based on structure-based molecular design. Our experimental results demonstrated that thirty-one of them possess remarkable tyrosinase inhibitory activities with IC₅₀ value below 1μM, and 5a, 6e, 6g and 6t did not display any toxicity to 293T cell line at the concentration of 1000μmol/L. According to the inhibitory activities, several compounds were selected for detail investigation on the structure–activity relationships (SARs), mechanisms of enzyme inhibition, inhibitory kinetics and cytotoxicity. In particular, the interaction between the selected inhibitors and the active center of tyrosinase was considered and discussed in detail based on their structural characteristics. Taken together, the results presented here demonstrated that the newly designed compounds are promising candidates for the treatment of tyrosinase-related disorders and further development of them may have significant contribution in biomedical science.

Full text of DOI:10.1248/cpb.c19-00949

Vol. 68, No. 4
Chem. Pharm. Bull. 68, 369–379 (2020)
369
Regular Article
Design, Synthesis, Biological Evaluation and Inhibition Mechanism of
3-/4-Alkoxy Phenylethylidenethiosemicarbazides as New, Potent and Safe
Tyrosinase Inhibitors
Senchuan Song, Yuliang Mai, Huahong Shi, Bing Liao*, and Fei Wang*
Guangdong Research Institute of Petrochemical and Fine Chemical Engineering, Guangdong Academy of Sciences;
Guangzhou 510665, P. R. China.
Received October 26, 2019; accepted January 22, 2020
Tyrosinase plays important roles in many different disease related processes, and the development of
its inhibitors is particularly important in biotechnology. In this study, thirty-nine 3-/4-alkoxyphenylethyli-
denethiosemicarbazides were synthesized as novel tyrosinase inhibitors based on structure-based molecular
design. Our experimental results demonstrated that thirty-one of them possess remarkable tyrosinase inhibi-
tory activities with IC50 value below 1µM, and 5a, 6e, 6g and 6t did not display any toxicity to 293T cell line
at the concentration of 1000µmol/L. According to the inhibitory activities, several compounds were selected
for detail investigation on the structure–activity relationships (SARs), mechanisms of enzyme inhibition, in-
hibitory kinetics and cytotoxicity. In particular, the interaction between the selected inhibitors and the active
center of tyrosinase was considered and discussed in detail based on their structural characteristics. Taken
together, the results presented here demonstrated that the newly designed compounds are promising candi-
dates for the treatment of tyrosinase-related disorders and further development of them may have significant
contribution in biomedical science.
Key words thiosemicarbazone; tyrosinase inhibitor; structure–activity relationship; inhibition mechanism;
inhibitory kinetics; cytotoxicity
functional optimization has not been reported.
Although the crystallographic structure of tyrosinase re-
Introduction
Tyrosinase (EC 1.14.18.1) is a multifunctional oxygenase
widely distributed in nature, structurally belonging to the vealed that there is a lipophilic long-narrow gorge near to
type-3 copper protein family.1–3) It catalyzes the hydroxylation the binuclear copper active center,57–59) which may be related
of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and the enzyme inhibitory mechanism, and the specific mode of
the oxidation of L-DOPA to dopaquinone. Dopaquinone is the interaction between the thiosemicarbazone molecule
highly reactive and can be oxidized spontaneously to form with these two copper ions in the active center of tyrosinase
melanin, which is responsible for the color of mammal-skin has been reported,45) the detailed binding process of inhibi-
and -hair.4–8) Recent studies showed that tyrosinase is in- tors to these two copper ions is still not known. According
volved in the molting process of insects9–12) and the browning to the information provided in-house51–53) (Fig. 1) and from
of fruits and vegetables.13–15) More importantly, tyrosinase other groups,42,54–56) thirty-nine novel 3-/4-alkoxy substituted
also plays important roles in many human diseases, includ- phenylethylidenethiosemicarbazide derivatives bearing both
ing Parkinson's and other neurodegenerative diseases,16–18) a thiosemicarbazone moiety and a proper alkoxy lipophilic
and the formation of some dermatological disorders, such as group were designed and synthesized, and their inhibitory
hyperpigmentation, freckles, melasma, ephelide, and senile effects on mushroom tyrosinase were evaluated. Furthermore,
lentigines.19–21) Therefore, tyrosinase inhibitors have become the structure–activity relationships (SARs) were discussed,
increasingly important in food industry, agriculture, cosmet- and the inhibition mechanisms, the inhibitory kinetics and
ics, and biomedical science.
the cytotoxicity of selected compounds were investigated in
Over the past decade, many efforts have been spent on the present study. Based on the results, the interaction process
searching for new and effective tyrosinase inhibitors, and between the thiosemicarbazone molecule of the inhibitors and
a tremendously large number of naturally occurring and the two copper ions in active center of tyrosinase was specu-
synthetic tyrosinase inhibitors have been reported.22–35) Un- lated. We hope that this study can lead to the development of
fortunately, only few of them can be used in practice due highly potent and safe pharmacological agents for the treat-
to the lack of their individual activities or safety concerns. ment of tyrosinase-related disorders.
Therefore, more efforts are urgently needed to search and
develop new, potent and safe tyrosinase inhibitors with im-
Results and Discussion
proved therapeutic profiles. Among those reported tyrosinase
Synthesis The synthesis of 5a–m, 6a–v, 11 and 12a–c was
inhibitors, thiosemicarbazide derivatives occupied a particu- summarized in Chart 1, and the structures of the correspond-
larly prominent position36–56) because of their remarkable in- ing alkoxy substituent at the phenyl ring were given in Table
hibitory activities against tyrosinase. However, the structural 1. Briefly, the alkoxy-acetophenones 3a–m, 4a–n, 4p–v and
modification of 1-(1-phenyl-ethylidene)thiosemicarbazides for the alkoxy-benzaldehydes 9, 10a–c were synthesized through
*To whom correspondence should be addressed. e-mail: wangfei@gdcri.com; gdcri2019@163.com
© 2020 The Pharmaceutical Society of Japan

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Fig. 1. Comparison of the Content in Previous Work and in This Work and the Aim of This Work
Reagents and reaction conditions: (a) RX (X=Br or I), K2CO3, anhydrous acetone, RT, 4–8h; (b) thiosemicarbazide, anhydrous ethanol, acetic acid, 50–80°C, 3h.
Chart 1. Synthesis of 1-(Alkoxyphenyl)ethylidenethiosemicarbazide Compounds (5a–m and 6a–v) and 2-Alkoxybenzylidenehydrazine-1-carbothio-
amide Compounds (11 and 12a–c)
the alkylation procedure. Subsequently, the condensation of corresponding results were listed in Table 1. As predicted, all
the corresponding 3a–m, 4a–v, 9, 10a–c with thiosemicarba- the structured-based condensation products displayed effective
zide was carried out in anhydrous alcohol using acetic acid as tyrosinase inhibitory activities, which was in good agreement
catalyst to provide the desired target compounds 5a–m, 6a–v, with our speculation.
11 and 12a–c in good to excellent yields, and their structures
From Table 1, the following SARs results could be derived:
a) 5a–m, 6a–v, 11 and 12a–c, a total of thirty-nine com-
pounds were synthesized, and thirty-one of them showed more
potent tyrosinase inhibitory activities than the well-known
1
were characterized by MS, H-NMR, 13C-NMR spectra and
element analysis.
Biological Activity
Inhibitory Effects of 5a–m, 6a–v, 11 and 12a–c on tyrosinase inhibitor kojic acid with IC50 values below 1µM.
Mushroom Tyrosinase b) From Fig. 2 we find that for 1-phenylethylidenethiosemi-
1-(1-Phenylethylidene)thiosemicarbazide had a potent in- carbazide compounds, the introduction of hydroxy or amino
hibitory activity on mushroom tyrosinase with IC50 value of group could not promote their inhibitory activity comparing
0.34µM.45) Inspired by this and with the established three- with unsubstituted 1-(1-phenylethylidene)thiosemicarbazide
dimensional structure57–59) of tyrosinase in mind, the inhibi- (IC50 value, 0.34µM),45) indicating that hydrophilic group is
tory effects of our synthetic thiosemicarbazide derivatives, not favorable substituent, even is disadvantageous one, such
5a–m, 6a–v, 11 and 12a–c, on mushroom tyrosinase were as in 1-(2,4,6-tyihydroxyphenyl)ethylidenethiosemi-carbazide
investigated, in which the well-known tyrosinase inhibitor, (IC50 value, 22.0µM).45)
kojic acid, and its parental compounds acetophenone and thi-
c) Compared to unsubstituted 1-(1-phenylethylidene)-
osemicarbazide were employed as the standard references. The thiosemicarbazide, compounds 5l, 5m, 6b, 6f, 6r, 6s, 6t, 6u

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Table 1. StructuresandIC50 Valuesof1-(3-Alkoxyphenyl)ethylidenethiosemicarbazideCompounds(5a–m),1-(4-Alkoxyphenyl)ethylidenethiosemicarbazide
Compounds (6a–v), 2-(4-(3-Hydroxypropoxy)benzylidene)hydrazine-1-carbothioamide (11) and 2-(3-Alkoxy-benzylidene)hydrazine-1-carbothioamide
Compounds (12a–c)
Entry
Alkoxy group structure
IC50 (µM)
Entry
Alkoxy group structure
IC50 (µM)
5a
5b
5c
C2H5
n-C3H7
0.848
0.748
0.620
0.525
0.768
1.040
0.707
1.061
0.690
0.353
1.152
0.590
0.828
>100
>100
28.5
6f
6g
6h
6i
(CH2)3OH
n-C4H9
0.334
0.408
0.353
0.432
0.269
0.404
0.251
0.790
1.091
0.237
0.566
0.699
0.439
0.686
0.182
0.323
0.747
1.017
2.250
1.361
1.150
n-C4H9
iso-C4H9
5d
5e
(CH2)4Cl
(CH2)4Cl
n-C5H11
n-C5H11
6j
5f
iso-C5H11
n-C6H13
6k
6l
n-C6H13
5g
Cyclohexyl
n-C7H15
5h
5i
n-C8H17
6m
6n
6o
6p
6q
6r
CH2Ph
n-C8H17
5j
CH2PhBr-4
(CH2)3Ph
Phenyl
5k
5l
CH2PhBr-4
(CH2)3Ph
CH2CO2C2H5
CH2CO2H
(CH2)3CO2C2H5
(CH2)3CO2H
CH2CH(OC2H5)2
(CH2)3OH
C2H5
CH2CO2C2H5
(CH2)3CO2C2H5
Acetophenones
Thiosemicarbazide
Kojic acid
C2H5
5m
Ref.
Ref.
Ref.
6a
6s
6t
6u
6v
11
12a
12b
12c
0.461
0.260
0.365
0.424
0.564
6b
6c
C2H4OH
n-C3H7
6d
6e
CH2CH=CH2
CH2C≡CH
n-C3H7
n-C4H9
Fig. 3. Active Site Structure of Tyrosinase57–59)
be concluded that the type of the substituent chain (with or
without hydrophilic group at the top) has no obvious influence
on inhibitory activity.
d) We find from Fig. 2 that the introduction of the lipophilic
group, hydroxyl or amino, to phenyl ring could enhance the
inhibitory activity of thiosemicarbazone compounds, the rea-
son for this phenomenon can be suggested as follows.
The active site structure of tyrosinase was shown in Fig.
3.57–59) There is a lipophilic “narrow gorge” in the middle of
the center of the enzyme and two copper ions are in middle of
the “narrow gorge.” It was found that there is a cap covering
“narrow gorge” and the cap is consisted of 378 nucleotides60,61)
which are flexible and prevent inhibitor binding to the active
Fig. 2. Comparison of the Effect of Introducing Hydrophilic Groups on
the Inhibitory Activity of Target Products
and 6v, bearing a side chain with hydrophilic group at the top, site of tyrosinase.57,62)
all showed efficient inhibitory activities and their IC50 values
Thiosemicarbazone molecule needs to enter and pass
were of the same order of magnitude (IC50 values from 0.182 through the “narrow gorge” before combining with two cop-
to 0.828µM), showing that the type of hydrophilic group at per ions. As thiosemicarbazo group is hydrophilic and easy to
the top of the chain has no obvious influence on inhibitory bind to nucleotides through van der Waals’ force. When the
activity in the present investigation. Subsequently, comparing thiosemicarbazone molecule approaches the “nucleotide” cap,
the IC50 values of compounds 5l, 5m, 6b, 6f, 6r, 6s, 6t, 6u and thiosemicarbazo group might induce the “nucleotide” cap to
6v with that of compounds 5a–e, 6a, 6c–e and 6g–m, it could change its conformation, which could provide convenience for

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Fig. 4. Structures of 5a, 6a and Their Three-Dimension Structures75)
Fig. 6. SAR Analysis
From Fig. 4 it was found that the value of molecular sec-
tion height of 5a is greater than that of 6a (h1 >h2), which
made 6a more favorable than 5a to enter and pass through
the “narrow gorge” of tyrosinase, and then, to combine with
Fig. 5. Comparison of Structures and Inhibitory Activities of 1-Alkoxy-
phenylethylidene-thiosemicarbazide Compounds and 2-Alkoxybenzyl-
idenehydrazine-1-carbothioamide Compounds
thiosemicarbazone molecule to enter the gorge. Subsequently, these two copper ions in the center of tyrosinase. In addition,
after the gate of the “narrow gorge” opens, the lipophilic the difference of electronic effect of group at 3-positon or
characteristic of the other end of thiosemicarbazone molecule 4-positon of the phenyl ring on thiosemicarbazone group may
would promote thiosemicarbazone molecule to get into the also participate in and affect the inhibitory activity of these
“narrow gorge” and further, to push thiosemicarbazone mol- two compounds.
ecule forward to the activity center of tyrosinase to combine
with these two copper ions.
f) 1-Alkoxyphenylethylidenethiosemicarbazide compounds
exhibited higher inhibitory activity than 2-alkoxybenzylidene-
Therefore, the introduction of lipophilic group to the other hydrazine-1-carbothioamide compounds with about a two-fold
end of the thiosemicarbazo group is benefit to enhance the in- of IC50 value, which indicated that on the benzylidene group
hibitory activity, but the lipophilicity and hydrophilicity of the of the molecule of thiosemicarbazide compounds methyl can
two ends of thiosemicarbazone molecule should be properly enhance the activity more than hydrogen (Fig. 5). Therefore,
adjusted, if the lipophilicity or hydrophilicity is too large the methyl group on benzylidene unit is another important group
inhibitory activity of the molecule could be reduced.
to enhance inhibitory activity.
e) In general, the compounds bearing the same alkoxy
The above SAR results were summarized and illustrated in
group at position-4 of the phenyl ring displayed more pro- Fig. 6.
found tyrosinase inhibitory activities than the ones bearing it
Inhibition Mechanism of Selected Compounds 5a and 6a
at position-3 of the phenyl ring. For example, the IC50 value on Tyrosinase The inhibition mechanism of compounds 5a
of 5a was 0.848µM, but the IC50 value of 6a was 0.461µM and 6a against mushroom tyrosinase for the oxidation of L-
(Table 1). The IC50 value of 5m was 0.828µM and the IC50 DOPA was investigated and the relationship between enzyme
value of 6t was 0.182µM. The results implied that the position activity and concentration of compounds 5a and 6a were re-
of the alkoxy substituent influenced the tyrosinase inhibitory spectively shown in Fig. 7. The results demonstrated that the
activity, and in the investigation, the alkoxy group attached plots gave a family of straight lines which all passed through
at position-4 of the phenyl ring was more efficacious for the the origin point. The increase of the inhibitor concentration
inhibitory potency. It was consistent with previous reports by resulted in the decrease of the line slope, indicating that the
Kubo,63) Jimenez64) and our recent report,52) and this phenom- inhibitory action of compounds 5a and 6a on mushroom ty-
enon is illustrated as follows through Fig. 4.
rosinase were reversible.45,65–69)

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373
Fig. 7. Determination of the Inhibitory Effects of 5a (A) and 6a (B) on Mushroom Tyrosinase for the Oxidation of L-DOPA
The concentrations of compound 5a for curves 1–4 were 0.0, 0.5, 1.0 and 2.0µM, respectively. The concentrations of compound 6a for curves 1–4 were 0.0, 0.1, 0.5 and
1.0 µM, respectively.
Fig. 8. Determination of the Inhibitory Types of Selected Compounds 5a (A) and 6a (B) on Tyrosinase for the Oxidation of L-DOPA
The concentrations of 5a for curves 1–3 were 2.0, 1.0 and 0.5µM, respectively. The concentrations of 6a for curves 1–3 were 1.0, 0.5 and 0.1µM, respectively.
Inhibitory Types of Selected Compounds 5a and 6a on
Since all of the selected compounds didn’t show any obvi-
Tyrosinase To further insight into the inhibitory mechanism, ous growth inhibition effect and toxicity to the 293T cell line
the inhibitory types of selected compounds 5a and 6a against even at a high concentration (1000µmol/L), we concluded that
mushroom tyrosinase for the oxidation of
L
-DOPA was deter- the compounds designed and selected in this study have low
mined by the Lineweaver–Burk double reciprocal plots with three cytotoxicity. In consideration of the cytotoxicity and promis-
different concentrations of inhibitors 5a and 6a, respectively (Fig. ing inhibitory effects against tyrosinase, these compounds
8). The results indicated that the plots of 1/V versus 1/[S] gave should have a broad application potential in food industry, ag-
three straight lines with different slopes, and they intersected riculture and cosmetics for the treatment of tyrosinase-related
one another at the ordinate. The values of Vmax remained the same disorders.
and the values of Km increased with the increase of inhibitor
concentrations. The results demonstrated that compounds 5a and
6a were the competitive tyrosinase inhibitors and also suggested
that they could only bind with the free enzyme.3,47,52,64,70)
Conclusion
In summary, we developed for the first time thirty-nine
3-/4-alkoxy substituted phenylethylidenethiosemicarbazides as
Toxicity of Compounds 5a, 6e, 6g and 6t to 293T Cell new, potent and safe tyrosinase inhibitors. The results showed
Line The 293T cell line rarely express endogenous recep- that thirty-one structure-based compounds had remarkable
tors required for extracellular ligands, and is easy to transfect, inhibitory activities with IC50 value of lower than 1µM, and
which is a very common cell line to express endogenous among them compound 6t exhibited the highest inhibitory
genes. In order to verify the safeties of these thiosemicarba- activity with an IC50 value of 0.182µM. The SAR analysis
zone compounds, four compounds 5a, 6e, 6g and 6t, consid- indicated that: i) the type of alkoxy group has no obvious
ering the diversity of structure and inhibition activity, were influence on the activity; ii) methyl group on benzylidene
selected to test the cytotoxicity to the 293T cell line.71–74) As group can enhance inhibitory activities; iii) the position of
demonstrated in Fig. 9, the results showed that these com- the alkoxy group obviously influenced the activity, and in the
pounds had no obvious growth inhibitory effect on the 293T investigation, the alkoxy group attached at position-4 was op-
cells at the concentrations of 0, 31.25, 62.5, 125, 250, 500, timal. Moreover, the inhibition mechanism and the inhibitory
1000µmol/L.
kinetics of compounds 5a and 6a revealed that these com-

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Fig. 9. Results of Cytotoxicity 5a, 6e, 6g and 6t to 293T Cell Line
pounds exhibited such tyrosinase inhibitory effects by acting in Hertz. Mass spectra were obtained from VG ZAB-HS,
as a reversible and competitive inhibitor, and the process of LCMS-2010A or LCQ DECA XP spectrometer. Elemental
the interaction was considered deeply between inhibitors and analyses were performed on a Vario EL instrument and were
the active center of tyrosinase, based on the polarity charac- within 0.4% of the theoretical values. All commercially
teristics of the two ends of inhibitors and structural charac- available reagents and solvents were used without further puri-
teristics of the active site of tyrosinase. Besides, the selected fication. Mushroom tyrosinase (specific activity of the enzyme
compounds 5a, 6e, 6g and 6t did not display any toxicity to is 6680U/mg) and L-DOPA (L-3,4-dhydroxyphenylalanine)
the 293T cell line even at the concentration of 1000µmol/L. were purchased from Sigma Chemical Co.
Taken together, these results suggested that these compounds
Procedure for the Synthesis of Targeted Compounds
could serve as the promising candidates for the treatment of 5a–m, 6a–v, 11 and 12a–c (Chart 1) Into 50mL of anhy-
tyrosinase-related disorders. Further investigation of these drous acetone were added 10.0mmol of compound 1 or 2,
compounds using human tyrosinase and a human melanoma 13.0mmol of the corresponding alkoxy bromide or alkyl io-
cell line is being conduct, and the results will be reported in dide and 20.0mmol of K2CO3, the above mixture was stirred
due course.
at room temperature for 4–8h. After completion of the reac-
tion as indicated by TLC, the reaction mixture was filtered
and the solvent was removed by evaporation at vacuum to
Experimental
General Melting points were determined on a WRS-1B get crude products, followed by chromatography to provide
digital instrument without correction. NMR spectra were re- the pure intermediates 3a–m, 4a–n, 4p–v. Compounds 9 and
corded on a Varian Mercury-Plus 300 spectrometer in dimeth- 10a–c were prepared as the same method. Then, they respec-
yl sulfoxide (DMSO)-d6. All chemical shifts (δ) were quoted tively reacted with thiosemicarbazide in the presence of acetic
in parts per million and coupling constants (J) were given acid (0.5mL) at 50–80°C for 3h to deliver the desired prod-

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
375
ucts 5a–m, 6a–v, 11 and 12a–c.
J=8.1, 1.9Hz, 1H), 4.04 (t, J=6.6Hz, 2H), 2.29 (s, 3H),
2- (1- (3 -Et hox y phe nyl) et hyl ide ne)hyd r a z i ne -1- 1.88–1.71 (m, 1H), 1.65–1.57 (m, 2H), 0.93 (d, J=6.6Hz, 6H).
carbothioamide (5a)
13C-NMR (75MHz, DMSO-d6) δ: 178.8, 158.7, 147.9, 139.1,
Solid product, yield 81%, mp 152–153°C. 1H-NMR 129.2, 119.0, 115.3, 112.4, 65.8, 37.5, 24.5, 22.4, 14.2. ESI-MS
(300MHz, DMSO-d6) δ: 10.15 (s, 1H), 8.25 (s, 1H), 7.91 (s, m/z=280.3 [M+H]+. Anal. Calcd for C14H21N3OS:C, 60.18;
1H), 7.46–7.37 (m, 2H), 7.26 (t, J=7.9Hz, 1H), 6.95–6.89 (m, H, 7.58; N, 15.04. Found C, 60.48; H, 7.54; N, 15.10.
1H), 4.06 (q, J=6.9Hz, 2H), 2.27 (s, 3H), 1.32 (t, J=6.9Hz,
2- (1- (3 -Hexoxy phe nyl) et hyl ide ne)hyd r a z i ne -1-
3H). 13C-NMR (75MHz, DMSO-d6) δ: 178.8, 158.5, 147.8, carbothioamide (5g)
139.1, 129.2, 119.0, 115.3, 112.4, 63.0, 14.6, 14.2. Electrospray
Solid product, yield 82%, mp 102–103°C. 1H-NMR
ionization (ESI)-MS, m/z=238.2 [M+H]+. Anal. Calcd for (300MHz, DMSO-d6) δ: 10.19 (s, 1H), 8.29 (s, 1H), 7.94 (s,
C11H15N3OS: C, 55.67; H, 6.37; N, 17.71. Found C, 55.99; H, 1H), 7.48–7.49 (m, 2H), 7.28 (t, J=7.9Hz, 1H), 6.95 (dd,
6.39; N, 17.79.
J=8.1, 1.9Hz, 1H), 4.01 (t, J=6.4Hz, 2H), 2.29 (s, 3H),
2- (1- (3-Propoxy phenyl)ethylidene)hyd ra zi ne -1- 1.79–1.62 (m, 2H), 1.51–1.20 (m, 6H), 0.88 (t, J=6.9Hz, 3H).
carbothioamide (5b)
13C-NMR (75MHz, DMSO-d6) δ: 178.8, 158.7, 147.9, 139.1,
Solid product, yield 82%, mp 114–115°C. 1H-NMR 129.2, 119.0, 115.3, 112.4, 67.4, 31.0, 28.7, 25.2, 22.1, 14.2, 13.9.
(300MHz, DMSO-d6) δ: 10.23 (s, 1H), 8.33 (s, 1H), 7.98 (s, ESI-MS m/z=294.3 [M+H]+.
1H), 7.46 (dd, J=7.4, 1.1Hz, 2H), 7.29 (t, J=8.1Hz, 1H),
2-(1-(3-Oct yloxy phenyl)ethylidene)hydrazine-1-
6.98–6.92 (m, 1H), 3.97 (t, J=6.5Hz, 2H), 2.31 (s, 3H), carbothioamide (5h)
1.82–1.66 (m, 2H), 1.00 (t, J=7.4Hz, 3H). 13C-NMR (75MHz,
Solid product, yield 72%, mp 107–108°C. 1H-NMR
DMSO-d6) δ: 178.9, 158.7, 147.9, 139.1, 129.2, 119.0, 115.2, (300MHz, DMSO-d6) δ: 10.18 (s, 1H), 8.28 (s, 1H), 7.94 (s,
112.4, 68.9, 22.1, 14.2, 10.4. ESI-MS m/z=252.2 [M+H]+. 1H), 7.48–7.49 (m, 2H), 7.28 (t, J=7.9Hz, 1H), 6.95 (dd,
Anal. Calcd for C12H17N3OS: C, 57.34; H, 6.82; N, 16.72. J=8.1, 2.0Hz, 1H), 4.00 (t, J=6.4Hz, 2H), 2.28 (s, 3H),
Found C, 57.45; H, 6.78; N, 16.66.
1.79–1.62 (m, 2H), 1.51–1.17 (m, 10H), 0.86 (t, J=6.9Hz, 3H).
2- (1- (3 -But ox y phe nyl) et hyl ide ne)hyd r a z i ne -1- 13C-NMR (75MHz, DMSO-d6) δ: 178.8, 158.7, 147.9, 139.1,
carbothioamide (5c)
129.2, 119.0, 115.3, 112.4, 67.4, 31.2, 28.7, 28.7, 28.7, 25.6,
Solid product, yield 78%, mp 105–106°C. 1H-NMR 22.1, 14.2, 13.9. ESI-MS m/z=322.4 [M+H]+. Anal. Calcd for
(300MHz, DMSO-d6) δ: 10.14 (s, 1H), 8.24 (s, 1H), 7.91 (s, C17H27N3OS: C, 63.51; H, 8.47; N, 13.07. Found C, 63.62; H,
1H), 7.46–7.36 (m, 2H), 7.25 (t, J=7.9Hz, 1H), 6.97–6.89 (m, 8.40; N, 13.13.
1H), 4.00 (t, J=6.4Hz, 1H), 1.76–1.61 (m, 1H), 1.50–1.37 (m,
2-(1-(3-Benzyloxyphenyl)ethylidene)hydrazine-1-
1H), 0.92 (t, J=7.3Hz, 1H). 13C-NMR (75MHz, DMSO-d6) δ: carbothioamide (5i)
178.8, 158.7, 148.0, 139.1, 129.2, 119.0, 115.3, 112.40, 7.1, 30.8,
Solid product, yield 86%, mp 143–144°C. 1H-NMR
18.7, 14.2, 13.7. ESI-MS m/z=266.2 [M+H]+. Anal. Calcd for (300MHz, DMSO-d6) δ: 10.21 (s, 1H), 8.32 (s, 1H), 7.93
C13H19N3OS: C, 58.84; H, 7.22; N, 15.83. Found C, 59.06; H, (s, 1H), 7.57–7.54 (m, 1H), 7.50–7.29 (m, 7H), 7.04 (dd,
7.18; N, 15.86.
2-(1-(3-(4-Chlorobutoxy)phenyl)ethylidene)hydrazine-1- (75MHz, DMSO-d6) δ: 178.9, 158.3, 147.7, 139.1, 137.1, 129.3,
carbothioamide (5d) 128.4, 127.8, 127.7, 119.4, 115.8, 112.6, 69.3, 14.2. ESI-MS
J=8.0, 2.1Hz, 1H), 5.17 (s, 2H), 2.29 (s, 3H). 13C-NMR
Solid product, yield 74%, mp 114–115°C. 1H-NMR m/z=300.3 [M+H]+.
(300MHz, DMSO-d6) δ: 10.15 (s, 1H), 8.26 (s, 1H), 7.91 (s,
2-(1-(3-((4-Bromobenzyl)oxy)phenyl)ethylidene)-
1H), 7.43 (d, J=8.9Hz, 2H), 7.26 (t, J=7.9Hz, 1H), 6.93 (d, hydrazine-1-carbothioamide (5j)
J=8.1Hz, 1H), 4.04 (t, J=5.3Hz, 2H), 3.70 (t, J=5.7Hz,
Solid product, yield 83%, mp 163–164°C. 1H-NMR
2H), 2.27 (s, 3H), 1.99–1.75 (m, 4H). 13C-NMR (75MHz, (300MHz, DMSO-d6) δ: 10.28 (s, 1H), 8.40 (s, 1H), 8.00 (s,
DMSO-d6) δ: 178.8, 158.5, 147.9, 139.1, 129.2, 119.1, 115.4, 1H), 7.63–7.55 (m, 3H), 7.51–7.41 (m, 3H), 7.32 (t, J=8.0Hz,
112.4, 66.7, 5.2, 28.9, 26.13, 14.2. ESI-MS m/z=300.3 1H), 7.04 (dd, J=8.1, 2.0Hz, 1H), 5.16 (s, 2H), 2.32 (s, 3H).
[M+H]+. Anal. Calcd for C13H18ClN3OS: C, 52.08; H, 6.05; N, 13C-NMR (75MHz, DMSO-d6) δ: 176.4, 155.6, 145.2, 136.6,
14.02. Found C, 52.31; H, 6.08; N, 13.91.
134.0, 128.8, 127.3, 126.8, 118.5, 117.0, 113.3, 110.1, 66.0, 11.7.
2- (1- (3-Pentoxy phenyl)et hylidene)hyd ra zi ne -1- ESI-MS m/z=378.2 [M+H]+. Anal. Calcd for C16H16BrN3OS:
carbothioamide (5e)
C, 50.80; H, 4.26; N, 11.11. Found C, 51.08; H, 4.35; N, 10.90.
2-(1-(3-(3-Phenylpropoxy)phenyl)ethylidene)hydrazine-1-
Solid product, yield 78%, mp 103–104°C. 1H-NMR
(300MHz, DMSO-d6) δ: 10.23 (s, 1H), 8.33 (s, 1H), 7.97 (s, carbothioamide (5k)
1H), 7.46 (d, J=7.6Hz, 2H), 7.29 (t, J=7.9Hz, 1H), 6.99–6.90
Solid product, yield 81%, mp 143–144°C. 1H-NMR
(m, 1H), 4.00 (t, J=6.4Hz, 2H), 2.31 (s, 3H), 1.78–1.62 (m, (300MHz, DMSO-d6) δ: 10.21 (s, 1H), 8.31 (s, 1H), 7.96 (s,
2H), 1.47–1.25 (m, 4H), 0.90 (t, J=7.1Hz, 3H). 13C-NMR 1H), 7.49–7.43 (m, 2H), 7.36–7.13 (m, 6H), 7.02–6.91 (m,
(75MHz, DMSO-d6) δ: 177.20, 157.00, 146.17, 137.40, 127.52, 1H), 4.02 (t, J=6.3Hz, 2H), 2.83–2.69 (m, 2H), 2.30 (s, 3H),
117.28, 113.53, 110.74, 65.74, 26.76, 26.09, 20.25, 12.55, 12.22. 2.11–1.95 (m, 2H). 13C-NMR (75MHz, DMSO-d6) δ: 178.9,
ESI-MS m/z=280.3 [M+H]+. Anal. Calcd for C14H21N3OS: 158.6, 147.9, 141.4, 139.1, 129.3, 128.3, 125.8, 119.1, 115.3,
C, 60.18; H, 7.58; N, 15.04. Found C, 60.33; H, 7.47; N, 15.02.
2-(1-(3-iso-Pentoxyphenyl)ethylidene)hydrazine-1- Anal. Calcd for C18H21N3OS: C, 66.02; H, 6.46; N, 12.83.
112.5, 66.7, 31.5, 30.4, 14.2. ESI-MS m/z=328.3 [M+H]+.
carbothioamide (5f)
Found C, 66.16; H, 6.47; N, 12.73.
Ethyl 2-(3-(1-(2-Carbamothioylhydrazono)ethyl)phenoxy)-
Solid product, yield 79%, mp 120–121°C. 1H-NMR
(300MHz, DMSO-d6) δ: 10.19 (s, 1H), 8.28 (s, 1H), 7.94 (s, acetate (5l)
1H), 7.49–7.38 (m, 2H), 7.28 (t, J=7.9Hz, 1H), 6.96 (dd,
Solid product, yield 84%, mp 154–155°C. 1H-NMR

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Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
(300MHz, DMSO-d6) δ: 10.21 (s, 1H), 8.32 (s, 1H), 7.97 (s,
2-(1-(4-(Prop-2-yn-1-yloxy)phenyl)ethylidene)hydrazine-1-
1H), 7.55–7.42 (m, 2H), 7.34–7.27 (m, 1H), 7.02–6.91 (m, carbothioamide (6e)
1H), 4.86 (s, 2H), 4.17 (q, J=7.1Hz, 2H), 2.29 (s, 3H), 1.21
Solid product, yield 78%, mp 175–176°C. 1H-NMR
(t, J=7.1Hz, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 178.9, (300MHz, DMSO-d6) δ: 10.10 (s, 1H), 8.19 (s, 1H), 7.87 (d,
168.7, 157.6, 147.5, 139.1, 129.3, 119.8, 115.7, 112.1, 64.7, 60.6, J=8.0Hz, 3H), 6.95 (d, J=8.5Hz, 2H), 4.83 (s, 2H), 3.56
14.1, 14.0. ESI-MS m/z=296.2 [M+H]+. Anal. Calcd for (s, 1H), 2.26 (s, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 179.2,
C13H17N3O3S: C, 52.86; H, 5.80; N, 14.23. Found C, 53.08; H, 158.7, 148.3, 131.4, 128.7, 115.2, 79.8, 79.1, 56.3, 14.7. ESI-MS
5.80; N, 14.25.
2-(1-(3-(4-(Ethylperoxy)butoxy)phenyl)ethylidene)- H, 5.30; N, 16.99. Found C, 58.41; H, 5.35; N, 16.81.
hydrazine-1-carbothioamide (5m) 2-(1-(4-(3-Hydroxypropoxy)phenyl)ethylidene)hydrazine-1-
Solid product, yield 75%, mp 107–108°C. 1H-NMR carbothioamide (6f)
m/z=248.2 [M+H]+. Anal. Calcd for C12H13N3OS: C, 58.28;
(300MHz, DMSO-d6) δ: 10.20 (s, 1H), 8.30 (s, 1H), 7.94 (s,
Solid product, yield 78%, mp 176–177°C. 1H-NMR
1H), 7.45 (t, J=5.8Hz, 2H), 7.29 (t, J=7.9Hz, 1H), 6.95 (dd, (300MHz, DMSO-d6) δ: 10.07 (s, 1H), 8.17 (s, 1H), 7.84 (d,
J=8.1, 1.8Hz, 1H), 4.15–3.98 (m, 4H), 2.47 (t, J=7.3Hz, J=8.0Hz, 3H), 6.88 (d, J=8.4Hz, 2H), 4.54 (t, J=5.0Hz,
2H), 2.29 (s, 3H), 2.04–1.92 (m, 2H), 1.18 (t, J=7.1Hz, 1H), 4.04 (t, J=6.2Hz, 2H), 3.60–3.49 (m, 2H), 2.24 (s, 3H),
3H). 13C-NMR (75MHz, DMSO-d6) δ: 178.8, 172.6, 158.4, 1.91–1.79 (m, 2H). 13C-NMR (75MHz, DMSO-d6) δ: 179.1,
147.8, 139.1, 129.3, 119.2, 115.3, 112.4, 66.5, 59.9, 30.1, 24.2, 160.2, 148.3, 130.4, 128.6, 114.5, 65.1, 57.7, 32.5, 14.3. ESI-MS
14.2, 14.1. ESI-MS m/z=324.3 [M+H]+. Anal. Calcd for m/z=268.4 [M+H]+.
C15H21N3O3S: C, 55.71; H, 6.54; N, 12.99. Found C, 55.67; H,
6.51; N, 13.02.
2- (1- (4 -Et hox y phe nyl) et hyl ide ne)hyd r a z i ne -1-
carbothioamide (6a)
2- (1- (4 -But ox y phe nyl) et hyl ide ne)hyd r a z i ne -1-
carbothioamide (6g)
Solid product, yield 83%, mp 166–167°C. 1H-NMR
(300MHz, DMSO-d6) δ: 10.09 (s, 1H), 8.18 (s, 1H), 7.86 (s,
Solid product, yield 85%, mp 153–154°C. 1H-NMR 1H), 7.84 (d, J=8.8Hz, 2H), 6.88 (d, J=8.9Hz, 2H), 3.98
(300MHz, DMSO-d6) δ: 10.15 (s, 1H), 8.24 (s, 1H), 7.88 (d, (t, J=6.4Hz, 2H), 2.25 (s, 3H), 1.76–1.61 (m, 2H), 1.47–1.36
J=8.9Hz, 2H), 6.91 (d J=8.9Hz, 2H), 4.12–3.97 (m, 2H), (m, 2H), 0.92 (t, J=7.3Hz, 3H). 13C-NMR (75MHz, DMSO-
2.28 (s, 3H), 1.34 (t, J=6.8Hz, 3H). 13C-NMR (75MHz, d6) δ: 178.6, 160.0, 147.8, 129.8, 128.1, 114.0, 67.2, 30.7, 18.7,
DMSO-d6) δ: 178.6, 159.5, 147.8, 129.8, 128.1, 113.9, 63.1, 13.8, 13.7. ESI-MS m/z=266.2 [M+H]+. Anal. Calcd for
14.6, 13.8. ESI-MS m/z=238.2 [M+H]+. Anal. Calcd for C13H19N3OS: C, 58.84; H, 7.22; N, 15.83. Found C, 59.06; H,
C11H15N3OS: C, 55.67; H, 6.37; N, 17.71. Found C, 55.94; H, 7.18; N, 15.86.
6.39; N, 17.79.
2-(1-(4-(2-Hydroxyethoxy)phenyl)ethylidene)hydrazine-1- carbothioamide (6h)
2-(1-(4-iso-Butoxyphenyl)ethylidene)hydrazine-1-
carbothioamide (6b)
Solid product, yield 86%, mp 148–149°C. 1H-NMR
Solid product, yield 83%, mp 217–218°C. 1H-NMR (300MHz, DMSO-d6) δ: 10.07 (s, 1H), 8.17 (s, 1H), 8.86 (s,
(300MHz, DMSO-d6) δ: 10.09 (s, 1H), 8.18 (s, 1H), 7.86 (s, 1H), 7.84 (d, J=8.7Hz, 2H), 6.89 (d, J=8.8Hz, 2H), 3.76
1H), 7.84 (d, J=8.6Hz, 2H), 6.90 (d, J=8.7Hz, 2H), 4.86 (d, J=6.5Hz, 2H), 2.25 (s, 3H), 2.07–1.94 (m, 1H), 0.98
(t, J=5.4Hz, 1H), 4.00 (t, J=4.7Hz, 2H), 3.71 (dd, J=9.7, (d, J=6.7Hz, 6H). 13C-NMR (75MHz, DMSO-d6) δ: 179.3,
4.9Hz, 2H), 2.25 (s, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 160.4, 148.5, 130.6, 128.7, 114.8, 74.6, 28.5, 19.9, 14.7. ESI-MS
179.3, 160.3, 148.5, 130.6, 128.8, 114.8, 70.4, 60.4, 14.7. ESI- m/z=266.2 [M+H]+. Anal. Calcd for C13H19N3OS: C, 58.84;
MS m/z=254.3 [M+H]+. Anal. Calcd for C11H15N3OS: C, H, 7.22; N, 15.83. Found C, 59.14; H, 7.15; N, 15.84.
52.15; H, 5.97; N, 16.59. Found C, 52.43; H, 5.88; N, 16.46.
2-(1-(4-(4-Chlorobutoxy)phenyl)ethylidene)hydrazine-1-
2- (1- (4 -Propoxy phenyl)ethylidene)hyd ra zi ne -1- carbothioamide (6i)
carbothioamide (6c)
Solid product, yield 83%, mp 159–160°C. 1H-NMR
Solid product, yield 84%, mp 185–186°C. 1H-NMR (300MHz, DMSO-d6) δ: 10.15 (s, 1H), 8.26 (s, 1H), 7.91 (s,
(300MHz, DMSO-d6) δ: 10.17 (s, 1H), 8.26 (s, 1H), 7.96–7.83 1H), 7.43 (d, J=8.9Hz, 2H), 7.26 (t, J=7.9Hz, 1H), 6.93 (d,
(m, 3H), 6.92 (d, J=9.0Hz, 2H), 3.94 (t, J=6.5Hz, 2H), 2.28 J=8.1Hz, 1H), 4.04 (t, J=5.3Hz, 2H), 3.70 (t, J=5.7Hz,
(s, 3H), 1.82–1.65 (m, 2H), 0.98 (t, J=7.4Hz, 3H). 13C-NMR 2H), 2.27 (s, 3H), 1.99–1.75 (m, 4H). 13C-NMR (75MHz,
(75MHz, DMSO-d6) δ: 178.6, 159.6, 147.8, 129.8, 128.1, 114.0, DMSO-d6) δ: 178.8, 158.5, 147.9, 139.1, 129.2, 119.1, 115.4,
68.9, 22.0, 13.8, 10.3. ESI-MS m/z=252.2 [M+H]+, Anal. 112.4, 66.7, 45.2, 28.9, 26.1, 14.2. ESI-MS m/z=300.2
Calcd for C12H17N3OS: C, 57.34; H, 6.82; N, 16.72. Found C, [M+H]+. Anal. Calcd for C13H18ClN3OS: C, 52.08; H, 6.05; N,
57.46; H, 6.77; N, 16.64.
14.02. Found C, 52.47; H, 6.09; N, 13.69.
2-(1-(4-(Allyloxy)phenyl)ethylidene)hydrazine-1-
carbothioamide (6d)
2- (1- (4 -Pentoxy phenyl)et hylidene)hyd ra zi ne -1-
carbothioamide (6j)
Solid product, yield 88%, mp 157–158°C. 1H-NMR
Solid product, yield 82%, mp 122–123°C. 1H-NMR
(300MHz, DMSO-d6) δ: 10.16 (s, 1H), 8.25 (s, 1H), 7.89 (d, (300MHz, DMSO-d6) δ: 10.18 (s, 1H), 8.27 (s, 1H), 7.89 (d,
J=8.9Hz, 3H), 6.95 (d, J=8.8Hz, 2H), 6.11–6.00 (m, 1H), J=8.9Hz, 3H), 6.91 (d, J=9.0Hz, 2H), 3.96 (t, J=6.5Hz,
5.41 (d, J=15.9Hz, 1H), 5.27 (d, J=10.5Hz, 1H), 4.60 (d, 2H), 2.29 (s, 3H), 1.77–1.63 (m, 2H), 1.45–1.25 (m, 4H),
J=5.1Hz, 2H), 2.28 (s, 3H). 13C-NMR (75MHz, DMSO-d6) 0.89 (t, J=7.0Hz, 3H). 13C-NMR (75MHz, DMSO-d6) δ:
δ: 178.6, 159.1, 147.8, 133.5, 130.1, 128.1, 117.5, 114.2, 68.2, 176.2, 157.3, 145.4, 127.5, 125.7, 111.6, 65.1, 26.0, 25.3, 19.6,
13.8. ESI-MS m/z=250.2 [M+H]+.
11.5, 11.5. ESI-MS m/z=280.3 [M+H]+. Anal. Calcd for
C14H21N3OS: C, 60.18; H, 7.58; N, 15.04. Found C, 60.46; H,
7.60; N, 14.92.

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2- (1- (4 -Hexoxy phe nyl) et hyl ide ne)hyd r a z i ne -1-
carbothioamide (6k)
2-(1-(4-(3-Phenylpropoxy)phenyl)ethylidene)hydrazine-1-
carbothioamide (6q)
Solid product, yield 82%, mp 140–141°C. 1H-NMR
Solid product, yield 85%, mp 148–149°C. 1H-NMR
(300MHz, DMSO-d6) δ: 10.15 (s, 1H), 8.24 (s, 1H), 7.88 (d, (300MHz, DMSO-d6) δ: 10.10 (s, 1H), 8.19 (s, 1H), 7.85 (d,
J=8.8Hz, 3H), 6.91 (d, J=8.9Hz, 2H), 3.97 (t, J=6.4Hz, J=8.6Hz, 3H), 7.31–7.11 (m, 5H), 6.89 (d, J=8.8Hz, 2H),
2H), 2.27 (s, 3H), 1.78–1.63 (m, 2H), 1.49–1.23 (m, 7H), 0.87 3.97 (t, J=6.2Hz, 2H), 2.73 (t, J=7.6Hz, 2H), 2.25 (s, 3H),
(t, J=6.7Hz, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 178.6, 2.06–1.95 (m, 2H). 13C-NMR (75MHz, DMSO-d6) δ: 179.3,
159.6, 147.7, 129.8, 128.1, 113.9, 67.5, 31.0, 28.6, 25.2, 22.1, 160.2, 148.5, 142.0, 130.7, 129.0, 128.8, 126.5, 114.8, 67.6,
13.9, 13.8. ESI-MS m/z=294.3 [M+H]+.
32.3, 31.2, 14.7. ESI-MS m/z=328.3 [M+H]+. Anal. Calcd for
2-(1-(4-Cyclohexyloxyphenyl)ethylidene)hydrazine-1- C18H21N3OS: C, 66.02; H, 6.46; N, 12.83. Found C, 66.13; H,
carbothioamide (6l)
6.47; N, 12.78.
Solid product, yield 88%, mp 160–161°C. 1H-NMR
Ethyl 2-(4-(1-(2-Carbamothioylhydrazono)ethyl)phenoxy)-
(300MHz, DMSO-d6) δ: 10.09 (s, 1H), 8.18 (s, 1H), 7.84 (d, acetate (6r)
J=8.3Hz, 3H), 6.91 (d, J=8.6Hz, 2H), 4.40 (s, 1H), 2.28 (s,
Solid product, yield 81%, mp 156–157°C. 1H-NMR
3H), 2.00–1.85 (m, 2H), 1.80–1.65 (m, 2H), 1.60–1.22 (m, 6H). (300MHz, DMSO-d6) δ: 10.09 (s, 1H), 8.18 (s, 1H), 7.85 (d,
13C-NMR (75MHz, DMSO-d6) δ: 179.3, 159.0, 148.6, 130.5, J=8.7Hz, 3H), 6.89 (d, J=8.8Hz, 2H), 4.81 (s, 2H), 4.15 (q,
128.8, 116.0, 75.1, 32.1, 26.0, 23.9, 14.7. ESI-MS m/z=292.3 J=7.1Hz, 2H), 2.25 (s, 3H), 1.21 (t, J=7.1Hz, 3H). 13C-NMR
[M+H]+.
(75MHz, DMSO-d6) δ: 179.3, 169.1, 159.1, 148.3, 131.4, 128.7,
2-(1-(4-Heptyloxyphenyl)ethylidene)hydrazine-1- 114.8, 65.5, 61.5, 14.9, 14.7. ESI-MS m/z=296.2 [M+H]+.
carbothioamide (6m) Anal. Calcd for C13H17N3O3S:C, 52.86; H, 5.80; N, 14.23.
Solid product, yield 79%, mp 126–127°C. 1H-NMR Found C, 52.74; H, 5.67; N, 14.19.
(300MHz, DMSO-d6) δ: 10.16 (s, 1H), 8.25 (s, 1H), 7.88 (d,
2-(4-(1-(2-Carbamothioylhydrazono)ethyl)phenoxy)acetic
J=8.9Hz, 3H), 6.90 (d, J=9.0Hz, 2H), 3.97 (t, J=6.4Hz, Acid (6s)
2H), 2.27 (s, 3H), 1.77–1.63 (m, 2H), 1.46–1.18 (m, 8H),
Solid product, yield 91%, mp 197–198°C. 1H-NMR
0.87 (t, J=9.1Hz, 3H). 13C-NMR (75MHz, DMSO-d6) δ: (300MHz, DMSO-d6) δ: 12.97 (s, 1H), 10.10 (s, 1H), 8.18 (s,
178.58, 159.63, 147.71, 129.82, 128.04, 113.93, 67.45, 31.23, 1H), 7.85 (d, J=8.6Hz, 2H), 6.87 (d, J=8.7Hz, 2H), 4.70
28.64, 28.47, 25.47, 22.05, 13.89, 13.79. ESI-MS m/z=308.3 (s, 2H), 2.25 (s, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 179.3,
[M+H]+. Anal. Calcd for C16H25N3OS: C, 62.50; H, 8.20; N, 170.6, 159.3, 148.4, 131.2, 128.7, 114.8, 65.3, 14.7. ESI-MS
13.67. FoundC, 62.43; H, 8.10; N, 13.48.
m/z=266.1 [M-H]−.
2-(1-(4 -Oct yloxy phenyl)ethylidene)hydrazine-1-
carbothioamide (6n)
Ethyl 4-(4-(1-(2-Carbamothioylhydrazono)ethyl)phenoxy)-
butanoate (6t)
Solid product, yield 77%, mp 127–128°C. 1H-NMR
Solid product, yield 71%, mp 129–130°C. 1H-NMR
(300MHz, DMSO-d6) δ: 10.08 (s, 1H), 8.18 (s, 1H), 7.83 (d, (300MHz, DMSO-d6) δ: 10.09 (s, 1H), 8.18 (s, 1H), 7.84 (d,
J=8.9Hz, 3H), 6.87 (d, J=8.9Hz, 2H), 3.95 (t, J=6.4Hz, J=8.5Hz, 3H), 6.88 (d, J=8.6Hz, 2H), 4.11–3.94 (m, 4H),
2H), 2.25 (s, 3H), 1.76–1.64 (m, 2H), 1.39–1.25 (m, 10H), 0.83 2.44 (t, J=7.2Hz, 2H), 2.25 (s, 3H), 2.04–1.89 (m, 2H), 1.16
(d, J=6.8Hz, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 179.2, (t, J=7.1Hz, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 179.2,
160.3, 148.4, 130.5, 128.7, 114.7, 68.3, 32.1, 29.6, 29.5, 26.4, 173.1, 160.0, 148.4, 130.7, 128.8, 114.7, 67.3, 60.6, 31.0, 25.0,
22.9, 14.8, 14.7. ESI-MS m/z=322.4 [M+H]+. Anal. Calcd for 15.0, 14.7. ESI-MS m/z=324.3 [M+H]+.
C17H27N3OS: C, 63.51; H, 8.47; N, 13.07. Found C, 63.44; H,
8.38; N, 12.95.
2- (1- (4 -Phenoxy phenyl)ethylidene)hyd razi ne-1-
carbothioamide (6o)
4-(4-(1-(2-Carbamothioylhydrazono)ethyl)phenoxy)butanoic
Acid (6u)
Solid product, yield 89%, mp 197–198°C. 1H-NMR
(300MHz, DMSO-d6) δ: 12.12 (s, 1H), 10.08 (s, 1H), 8.17
Solid product, yield 82%, mp 171–172°C. 1H-NMR (s, 1H), 7.84 (d, J=8.3Hz, 2H), 6.89 (d, J=8.5Hz, 2H),
(300MHz, DMSO-d6) δ: 10.15 (s, 1H), 8.22 (s, 1H), 7.92 4.00 (t, J=6.2Hz, 2H), 2.38 (t, J=7.2Hz, 2H), 2.25 (s, 3H),
(d, J=8.9Hz, 2H), 7.88 (s, 1H), 7.43–7.30 (m, 2H), 7.13 (t, 2.02–1.85 (m, 2H). 13C-NMR (75MHz, DMSO-d6) δ: 179.2,
J=7.4Hz, 1H), 7.03–6.98 (m, 2H), 6.93 (d, J=8.9Hz, 2H), 174.6, 160.1, 148.5, 130.7, 128.8, 114.7, 67.4, 30.9, 25.0, 14.7.
2.25 (s, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 178.8, 157.7, ESI-MS m/z=294.1 [M-H]−.
156.1, 147.2, 132.7, 130.1, 128.5, 123.8, 119.0, 117.8, 13.9. ESI-
2-(1-(4-(2,2-Diethoxyethoxy)phenyl)ethylidene)hydrazine-1-
carbothioamide (6v)
MS m/z=286.4 [M+H]+.
2-(1-(4-((4-Bromobenzyl)oxy)phenyl)ethylidene)-
hydrazine-1-carbothioamide (6p)
Solid product, yield 75%, mp 136–137°C. 1H-NMR
(300MHz, DMSO-d6) δ: 10.09 (s, 1H), 8.18 (s, 1H), 7.85 (d,
Solid product, yield 81%, mp 197–198°C. 1H-NMR J=8.9Hz, 3H), 6.92 (d, J=9.0Hz, 2H), 4.79 (t, J=5.1Hz,
(300MHz, DMSO-d6) δ: 10.08 (s, 1H), 8.17 (s, 1H), 7.86 (d, 1H), 3.98 (d, J=5.1Hz, 2H), 3.75–3.47 (m, 4H), 2.25 (s,
J=8.8Hz, 3H), 7.57 (d, J=8.3Hz, 2H), 7.39 (d, J=8.4Hz, 3H), 1.14 (t, J=7.0Hz, 6H). 13C-NMR (75MHz, DMSO-d6)
2H), 6.96 (d, J=8.9Hz, 2H), 5.12 (s, 2H), 2.24 (s, 3H). δ: 179.2, 159.8, 148.3, 131.0, 128.8, 114.85, 100.6, 68.9, 62.7,
13C-NMR (75MHz, DMSO-d6) δ: 178.6, 159.0, 147.7, 136.4, 16.2, 14.7. ESI-MS m/z=326.3 [M+H]+. Anal. Calcd for
131.3, 130.4, 129.8, 128.13, 120.9, 114.4, 68.4, 13.8. ESI-MS C15H23N3O3S: C, 55.36; H, 7.12; N, 12.91. Found C, 55.66; H,
m/z=378.2 [M+H]+. Anal. Calcd for C16H16BrN3OS: C, 7.14; N, 13.24.
50.80; H, 4.26; N, 11.11. Found C, 50.61; H, 4.33; N, 10.83.
2-(4-(3-Hydroxypropoxy)benzylidene)hydrazine-1-
carbothioamide (11)
Yellowish solid product, yield 85%, mp 172–173°C.

378
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
1H-NMR (300MHz, DMSO-d6) δ: 11.34 (s, 1H), 8.14 (s, 1H), (37°C, 5% CO2); 10µL of different-concentration solution of
8.02 (s, 1H), 7.94 (s, 1H), 7.74 (d, J=8.8Hz, 1H), 6.96 (d, 5a was added to each cell line tube and the final concentra-
J=8.8Hz, 1H), 4.60 (t, J=5.1Hz, 1H), 4.08 (t, J=6.4Hz, tion of 5a was respectively kept in 31.25, 62.5, 125, 250, 500,
2H), 3.58 (t, J=6.1Hz, 2H), 1.88-1.91 (m, 2H). 13C-NMR 1000µmol/L; The culture plate was incubated for 24h in the
(75MHz, DMSO-d6) δ: 177.5, 160.1, 142.3, 128.9, 126.52, incubator, then 10µL of CCK-8 solution was added into each
114.5, 64.7, 57.2, 32.0. ESI-MS m/z=254.3 [M+1]+. Anal. tube in 96-hole plate; After the culture plate was incubated
Calcd for C11H15N3O2S: C, 52.16; H, 5.97. Found C, 52.30; H, for 4h in the incubator, the absorbance of the mixture in each
6.01.
2-(3-Ethyoxybenzylidene)hydrazine-1-carbothioamide (12a)
tube at 450nm was determined using micro-plate reader.
The cell line tube to which compound 5a was not injected
Solid product, yield 92%, mp 165–166°C. 1H-NMR was used as reference and the cell-proliferation number of this
(300MHz, DMSO-d6) δ: 11.44 (s, 1H), 8.27 (d, J=19.3Hz, cell line was assumed “1.” At this time the cell proliferation
1H), 8.09 (s, 1H), 8.03 (s, 1H), 7.45 (s, 1H), 7.38–7.17 (m, 2H), number for each cell line tube was determined as 1.012, 1.086,
7.02–6.90 (m, 1H), 4.07 (q, J=7.0Hz, 2H), 1.41–1.26 (m, 3H). 1.086, 1.027, 1.119, 1.014. With the same method, we also
13C-NMR (75MHz, DMSO-d6) δ: 177.9, 158.8, 142.2, 135.5, determined the cytotoxicities of compounds 6e, 6g and 6t to
129.7, 120.5, 116.6, 111.4, 63.1, 14.6. ESI-MS m/z=224.2 293T cell line.
[M+1]+.
2-(3-Propoxybenzylidene)hydrazine-1-carbothioamide (12b)
Acknowledgments We thank the GDAS’ Project of Sci-
Solid product, yield 89%. mp 161–162°C. 1H-NMR ence and Technology Development (2018GDASCX-1014 and
(300MHz, DMSO-d6) δ: 11.42 (s, 1H), 8.21 (s, 1H), 8.08 (s, 2018GDASCX-0807), the Project of Tianhe District, Guang-
1H), 8.01 (s, 1H), 7.44 (s, 1H), 7.34–7.20 (m, 2H), 6.99–6.92 zhou for Innovation and Entrepreneurship Leading Talents,
(m, 1H), 3.97 (t, J=6.5Hz, 2H), 1.81–1.65 (m, 2H), 0.99 (t, and the Project of Foshan Science and Technology Innovation
J=7.4Hz, 3H). 13C-NMR (75MHz, DMSO-d6) δ: 177.9, 159.0, for financial support of this study.
142.2, 135.5, 129.7, 120.5, 116.6, 111.5, 69.0, 22.0, 10.4. ESI-
MS m/z=238.2 [M+1]+.
Conflict of Interest The authors declare no conflict of
interest.
2-(3-Butoxybenzylidene)hydrazine-1-carbothioamide (12c)
Solid product, yield 86%, mp 151–152°C. 1H-NMR
(300MHz, DMSO-d6) δ: 11.35 (s, 1H), 8.18 (s, 1H), 8.04
(s, 1H), 7.97 (s, 1H), 7.41 (s, 1H), 7.3–7.17 (m, 2H), 6.92 (d,
J=9.1Hz, 1H), 3.99 (t, J=6.4Hz, 2H), 1.76–1.61 (m, 2H),
1.51–1.36 (m, 2H), 0.93 (t, J=7.3Hz, 3H). 13C-NMR (75MHz,
DMSO-d6) δ: 177.9, 159.0, 142.2, 135.5, 129.6, 120.5, 116.6,
111.5, 67.2, 30.8, 18.7, 13.7. Anal. Calcd for C12H17N3OS: C,
57.34; H, 6.82. Found C, 57.30; H, 6.86.
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