Study on the design, synthesis and structure-activity relationships of new thiosemicarbazone compounds as tyrosinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.08.033

52 Structure-based thiosemicarbazone compounds bearing various substituted-lipophilic part, including substituted-benzaldehyde, substituted-phenylalkan-1-one and their biphenyl-type thiosemicarbazone analogs, were designed, synthesized and evaluated as new tyrosinase inhibitors. The results demonstrated that 22 compounds have potent inhibitory activities against tyrosinase with the IC₅₀ value of lower than 1.0 μM. On the basis of the obtained experimental data, the structure-activity relationships (SARs) were rationally derived. Besides, the inhibition mechanism and the inhibitory kinetics of selected compounds 3d and 6e were investigated, revealing that such type of compounds were belonged to the reversible and competitive tyrosinase inhibitors. To verify the safety of these developed thiosemicarbazone compounds, four randomly selected compounds 3d, 4e, 6a and 9a were also tested in 293T cell line for the evaluation of the cytotoxicity. Interestingly, all these compounds almost did not perform any toxicity to 293T cells even at a high concentration of 1000 μmol/L. Taken together, these results suggested that such compounds could serve as the highly efficient and more safe candidates for the treatment of tyrosinase-related disorders.

Full text of DOI:10.1016/j.ejmech.2017.08.033

Accepted Manuscript
Study on the design, synthesis and structure-activity relationships of new
thiosemicarbazone compounds as tyrosinase inhibitors
Senchuan Song, Ao You, Zhiyong Chen, Guoxun Zhu, Huan Wen, Huacan Song, Wei
Yi
PII:
S0223-5234(17)30636-0
10.1016/j.ejmech.2017.08.033
EJMECH 9677
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 April 2017
Revised Date: 27 July 2017
Accepted Date: 14 August 2017
Please cite this article as: S. Song, A. You, Z. Chen, G. Zhu, H. Wen, H. Song, W. Yi, Study on
the design, synthesis and structure-activity relationships of new thiosemicarbazone compounds
as tyrosinase inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.033.
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ACCEPTED MANUSCRIPT
"Developing new thiosemicarbazone compounds as potent and safe tyrosinase inhibitors"
R''
N
IC50 (µM)
3d 0.339; 3i 0.339; 5a 0.325;
5b 0.352; 6a 0.236; 6b 0.177;
6c 0.142; 6d 0.394; 7a 0.232;
7b 0.278; 9a 0.278.
R'
NH2
S
H
N
Middle-phenyl
Toxicity to 293T cell
3d, 4e, 6a and 9a did not
show toxicities even at a
high concentration of
1000 µmol / L.

ACCEPTED MANUSCRIPT
Study on the design, synthesis and structure-activity
relationships of new thiosemicarbazone compounds
as tyrosinase inhibitors
Senchuan Songa,c, Ao Youa, Zhiyong Chenc*, Guoxun Zhua, Huan Wend,
Huacan Songa,*, Wei Yi a,b*
a School of Chemical Engineering and Technology, Sun Yat-sen University, 135 Xin Gang West
Road, Guangzhou 510275, P.R. China
b Key Laboratory of Molecular Clinical Pharmacology & Fifth Affiliated Hospital, Guangzhou
Medical University, Guangzhou, Guangdong 511436, P. R. China
c China National Analysis Center, Guangzhou 510070, P. R. China
dGuangdong Huizhou Testing Center for Petroleum Product Quality, Huizhou 516003, P. R.
China
*Corresponding Author.
E-mail: yiwei2@mail2.sysu.edu.cn, songhc2007@163.com, or chenzh@fenxi.com.cn
Abstract
52
Structure-based
thiosemicarbazone
compounds
bearing
various
substituted-lipophilic
part, including
substituted-benzaldehyde,
substituted-phenylalkan-1-one and their biphenyl-type thiosemicarbazone analogs,
were designed, synthesized and evaluated as new tyrosinase inhibitors. The results
demonstrated that 22 compounds have potent inhibitory activities against tyrosinase
with the IC50 value of lower than 1.0 µM. On the basis of the obtained experimental
data, the structure-activity relationships (SARs) were rationally derived. Besides, the
inhibition mechanism and the inhibitory kinetics of selected compounds 3d and 6e
were investigated, revealing that such type of compounds were belonged to the
reversible and competitive tyrosinase inhibitors. To verify the safety of these
developed thiosemicarbazone compounds, four randomly selected compounds 3d, 4e,
6a and 9a were also tested in 293T cell line for the evaluation of the cytotoxicity.
Interestingly, all these compounds almost did not perform any toxicity to 293T cells
even at a high concentration of 1000 µmol /L. Taken together, these results suggested
that such compounds could serve as the highly efficient and more safe candidates for
the treatment of tyrosinase-related disorders.
Keywords: Thiosemicarbazone compounds; Tyrosinase inhibitors; SARs; Inhibition
mechanism; Inhibitory kinetics; Cytotoxicity.

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1. Introduction
Tyrosinase is the key enzyme in melanin biosynthesis, which plays an important
role in multiple physiological and pathological pathways. For example, it can control
the dopamine toxicity, and consequently, influencing the inducion and development
process of many important neurodegenerative diseases, such as Parkinson's disease
[1,2]. Recent studies also showed that its activity is closely related to the incidence of
melanoma [3]. Moreover, tyrosinase can promote the development and defense
function of insect and plays a vital role in melanin formation, wound healing and
resistance to parasites and skin keratinization physiological processes [4-7].
Addationally, the browning of fruits and vegetables has a direct relationship with
tyrosinase-involved activity [8-10]. Therefore, the research and development of
highly effective and more safe tyrosinase inhibitors has become increasingly
important in medication, cosmetic industry, agriculture as well as food industry.
Fig. 1 (1) Crystal structure of tyrosinase; (2) Enlarged picture for active structure of tyrosinase.
Recently, the X-ray overall structure of tyrosinase have been respectively
determined or predicted by the groups of Sugiyama, Vaya, Fishman, and Dijkstra (Fig.
1.1) [11-14]. As shown in Fig. 1.2, the active structure of tyrosinase contains a narrow
“gap” and two center copper ions, which were separated by the “gap” and respectively
coordinated with three different amino acids. In the reduced state, the two copper ions
were linked by one oxygen atom; but in the oxidized state, these two copper ions were
linked by two oxygen atoms, such a state is easy to offer one oxygen atom to oxidize
substrate L-dopamine. Further analysis indicated that this narrow “gap” is lipophilic.
These information give a meaningful direction for the structure-based design and
development of more effective tyrosinase inhibitors.

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It was reported that, 4-alkoxy-/4-acyloxyphenylethylenethiosemicarbazone
analogues (Fig. 2.1) [15], and 1-(1-(4-amidophenyl)ethylidene)thiosemicarbazone
analogues (Fig. 2.2) [16] could perform high activities against tyrosinase. In the view
of their molecular structures, we found that the thiosemicarbazone group is core unit
for these two kinds of compounds.
Fig. 2. Structures of 4-alkoxy- and 4-acyloxyphenylethylenethiosemicarbazone analogues (1)
and 1-(1-(4-amidophenyl)ethylidene)thiosemicarbazone analogues (2)
By analyzing these reported representative structures of thiosemicarbazone
compounds, in general, they could be divided into two parts (Fig. 3): the hydrophilic
part (thiosemicarbazone-group part) and the lipophilic part (the –R part).
N
R
NH
Lipophilic
part
Hydrophilic
part
S
R
H2N
Fig. 3 Hydrophilic part and the lipophilic part of thiosemicarbazone compounds
Since the narrow “gap” of tyrosinase is lipophilic, the increase of the
hydrophobicity of the -R part of the inhibitor molecules would be favorable for them
to entry the narrow “gap” of tyrosinase, and consequently, increasing the chance of
thiosemicarbazone part in inhibitor molecules to combine with cooper ion enter.
Based on the structural and biochemical information of tyrosinase and in continuation
of our interest in developing the novel benzylidenethiosemicarbazides as the potent
tyrosinase inhibitors [15, 16], we herein designed and synthesized several new series
of
substituted-lipophilic
substituted-phenylalkan-1-one and their biphenyl-type thiosemicarbazone analogues
structure-based
thiosemicarbazone
compounds
bearing
various
part, including
substituted-benzaldehyde,

ACCEPTED MANUSCRIPT
(Fig. 4), with the aim to find new tyrosinase inhibitors with more efficient inhibitory
activities and lower toxicities.
N
NH
NH
N
R'
R'
S
S
R'' H2N
R''
H2N
B middle-phenyl
A
4-Substituted phenylalkan-1-one
thiosemicarbazone compounds
2-(1-(4'-Substituted-[1,1'-biphenyl]-4-yl)-
ethylidene)hydrazine-1-carbothioamide compounds
Fig. 4. The basic framework of tyrosinase inhibitors involved in this study
2. Results and Discussion
H
N
NH2
N
S
R2
R1
3a-j
O
S
O
R4COCl
R2
R3
N
R3
N
NH2
N
R1
R3
R4
H
R4
4a'-p'
4a-p
S
O
N
NH2
R5OH
R5O2C(CH2)n
H
R5O2C(CH2)n
O
S
CH3
n = 1, 5a-d; n = 2, 6a-d; n = 3, 7a-c
CH3
H2NHN
NH2
n = 1, 2, 3
CH3
n = 1, 2, 3
OH
n = 1, 5a'-d'; n = 2, 6a'-d'; n = 3, 7a'-c'
R6COO
R6COO
R6COO
R6COCl
R7X
CH3
Acetic anhydride
AlCl3
S
8a'-e'
CH3
R7O
8a-e
8g-j
R7O
N
N
NH2
R7O
O
H
CH3
CO2H
COOR8
S
8g'-j'
CH3
COOR8
N
COOR8
N
NH2
R8OH
H
O
CH3
S
9a-d
9a'-d'
CH3
N
8f
8a
9a
N
NH2
H
(a) NaOH
(b) HCl
O
9e
Scheme 1 Synthetic method and structures of target compounds
2.1. Chemistry
The synthetic route of target compounds was showed in Scheme 1. Briefly, to
probe the effect of the nature, the chain length and the chain space volume of
lipophilic moiety on tyrosinase inhibitory activity, compounds 3a-j were firstly
synthesized by condensation reaction of the corresponding and commercially
available substituted benzaldehydes or substituted acetophenones with
thiosemicarbazide. However, for the synthesis of compounds 4a-p, substituted
acetophenones (4a’-p’) were firstly prepared via Friedel-Crafts reaction of the
commercial available alkylbenzene with acyl chloride. Subsequently, treatment of
4a’-p’and thiosemicarbazide gave target products 4a-p in good yield by following the

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synthetic method of compounds 3a-j. Similarly, alkyl phenylacetates, alkyl
3-phenylpropanoates or alkyl 4-phenylbutanoates were acetylated to offer the
corresponding 4-acetylatedphenylalkanoate ester intermediates (5a’-d’, 6a’-d’ and
7a’-c’), which further reacted with thiosemicarbazide to provide target compounds
5a-d, 6a-d and 7a-c with good yield.
To explore the inhibitory effect of biphenyl-type thiosemicarbazone compounds
and define the importance of middle-phenyl moiety, 4'-acetylbiphenyl-4-carboxylic
acid alkyl ester (8a’-e’) were synthesized by esterification with acyl chloride and then
acetylation with acetic anhydride via Friedel-Crafts reaction. 8a’-e’ reacted with
thiosemicarbazide giving 8a-e. The another kind of biphenyl-type thiosemicarbazone
analogues, 8g-j were also synthesized successfully though an alkylation, acetylation
and condensation three-step sequence in which 8g’-j’ were employed as the
intermediates. Finally, 9a-d were smoothly synthesized via an etherification,
acetylation
and
condensation
process
by
using
alkyl
(4-'acetyl-4-[1,1'-biphenly])acetaes 9a’-d’ as the intermediates. The hydrolysis of 8a
and 9a provided 8f and 9e, respectively.
2.2. Biological activity of the target compounds
The inhibitory activity of all target compounds were evaluated on tyrosinase
using kojic acid as reference according to the reported protocol [15, 17], and the
activity data as IC50 value was presented in Table 1. The results showed that, among
these target compounds, 22 compounds performed higher activities with IC50 value of
lower than 1.0 µM. Especially, compounds 6a-c, 7a, 7b and 9a exhibited more potent
tyrosinase inhibitory activities (IC50 value < 0.3 µM) than our previous reported
4-alkoxy- and 4-acyloxyphenyl-ethylenethiosemicarbazone analogues [15] and
4-alkoxy- and 4-acyloxy-phenylethylenethiosemicarbazone analogues [16], revealing
that they were great potential tyrosinase inhibitors.

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COOR8
S
R7O
R6COO
H
N
S
NH2
NH2
R3
N
N
N
NH2
N
H
N
S
H
CH3
n = 1, 5a-d; n = 2, 6a-d; n = 3, 7a-c
CH3
R2
CH3
CH3
R1
R4
4a-p
S
S
S
8g-j
8a-f
3a-j
9a-e
N
N
N
N
NH2
N
NH2
N
NH2
H
H
H
Samples
3a
R1
R2
IC50 (µM) Samples
R1
R2
IC50 (µM)
1.912
4-propyl-iso
-CH=CH-CH=CH-
(2-, 3-)
methyl
H
2.83
3f
3-methyl
-CH2CH2CH2CH2-
(2-, 3-)
methyl
2.382
3b
methyl
1.982
3g
3c
2,3-dimethyl
4-butyl
methyl
methyl
2-methyl-2-
hydroxyethyl
R4
0.448
0.339
3h
3i
4-phenlyl
H
methyl
H
2.781
0.384
3d
4-phenlyl
-butyl-ter
4-styryl
3e
>200
3j
3.101
R3
R3
R4
4a
4b
4c
4d
4e
4f
4-methyl
4-methyl
4-methyl
4-propyl-iso
propyl
35.35
115.15
>100
88.97
0.610
0.960
9.49
4i
4j
4-pentyl
methyl
methyl
methyl
ethyl
0.495
0.483
0.632
8.12
iso-propyl
benzyl
4-hexyl
4k
4l
4-cyclohexyl
iso-propyl
methyl
methyl
ethyl
4-cyclohexyl
4-(1-methylpropyl)
4m
4n
4o
4p
4-cyclohexyl
propyl
56.67
98.31
>100
>100
4-butyl-ter
4-cyclohexyl
iso-propyl
benzyl
benzyl
R5
4g
4h
4-butyl
4-cyclohexyl
4-butyl
propyl
28.81
4-H
n
2
n
1
1
1
1
2
2
R5
5a
5b
5c
5d
6a
6b
methyl
ethyl
0.325
0.358
0.458
0.441
0.236
0.177
6c
6d
7a
7b
7c
propyl
H
0.142
0.394
0.232
0.278
5.901
2
propyl
3
methyl
ethyl
butyl
3
methyl
ethyl
3
butyl
R6
R7
H
8a
8b
8c
8d
8e
/
/
/
/
/
acetyl
1.513
1.992
2.466
> 50
8f
8g
8h
8i
/
/
/
/
/
13.904
17.566
> 50
iso-propionyl
pivaloyl
benzoyl
4-methoxybenzolyl
R8
ethyl
propyl
butyl
pentyl
R8
> 50
> 50
8j
> 50
9a
9b
9c
/
/
/
methyl
ethyl
0.278
0.437
0.485
9d
9e
/
/
/
butyl
H
0.562
1.661
28.2
propyl
Kojic acid
/
Table 1 Structures and inhibitory activities of all target compounds

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2.2.1. Relationships between activity and structures of the thiosemicarbazone
compounds as tyrosinase inhibitors
The basic framework of structure-based thiosemicarbazone compounds involved in
this study were enumerated in Fig. 4. In combination with our obtained data, several
points for SARs analysis were discussed as follows.
2.2.1.1. The inhibitory effect of 4-alkylphenylalkan-1-one thiosemicarbazone
compounds
For substituted-benzaldehyde, substituted-phenylalkan-1-one thiosemicarbazone
compounds (Fig. 4.A), the effect of R’ and R” parts was mainly divided into three
aspects, including the nature, the chain length and the chain space volume.
(i) The inhibitory effect of the nature of R’: In general, 4-ester-alkylacetophenone
thiosemicarbazone compounds showed more potent activities than the corresponding
4-alkylacetophenone thiosemicarbazone compounds (for 4-ester-substituted
compounds: 5a-d, 6a-d and 7a-b with the range of IC50 values from 0.142 to 0.458
µM vs for 4-alkyl-substituted compounds 4e-f and 4i-k with the range of IC50 values
from 0.495 to 5.901 µM). The results indicated that the introduction of appropriate
ester group into position-4 of benzene ring is benificial to tyrosinase inhibitory
activity, which might be mainly due to form the hydrogen bonding interaction
between the oxygen atom and the amino acid residue of the lipophilic pocket of
tyrosinase.
(ii) The inhibitory effect of the chain length of R’: By analysis of the data form Table
1, we found the chain length of the R’ has no obvious influence on the inhibitory
activity (e.g. 3d (0.339 µM, R’ = n-C4H9) vs 4i (0.495 µM, R’ = n-C5H11) vs 4i (0.483
µM, R” = n-C6H13)). The similar conclusion was observed in 4-ester-substituted
compounds 5a (0.325 µM, R’ = CH2CO2CH3), 6a (0.236 µM, R’ = (CH2)2CO2CH3)
and 7a (0.232 µM, R’ = (CH2)3CO2CH3). However, when the chain length of R” is
more than 8 atoms, the activities of inhibitors decreased sharply (e.g. 7b (0.278 µM,
R’ = (CH2)3CO2C2H5) vs 7c (5.901 µM, R’ = (CH2)3CO2n-C4H9)), which is consistent
with our previous investigation that used 4’-/3’-alkoxylacetophenone
thiosemicarbazone compounds as tyrosinase inhibitors [15].
(iii) The inhibitory effect of the chain space volume of R’: As demonstrated in Table 1,

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we are able to found that compounds 3d (R’ = 4-n-butyl), 4e (R’ =
4-(1-methylpropyl)), 4k (R’ = 4-cyclohexyl) and 4f (R’ =4-butyl-ter) display different
inhibitory activities against tyrosinase, suggesting that the chain space volume of R”
plays an important role in determining the inhibitory activity, and in the present
investigation, with the increase of the space volume of alkyl group on 4-positon of
phenyl ring, the inhibitory activity of the corresponding thiosemicarbazone
compounds decreased generally.
(iv) The inhibitory effect of R’’: Our previous reports have demonstrated that
alkylactophenone thiosemicarbazone compounds have a more potent inhibitory
activity against tyrosinase than that of the corresponding alkylbenzaldehyde
thiosemicarbazone compounds. Inspired by this finding, herein a series of
thiosemicarbazone compounds bearing diverse R’’ group, such as methyl, ethyl,
propyl, iso-propyl, 2-hydroxy-2-methylethyl and benzyl, was designed, synthesized
and evaluated as new tyrosinase inhibitors. As listed in Table 1, compound 4k (R’’ =
CH3) showed a potent tyrosinase inhibitory activity with the IC50 value of 0.632 µM.
Changing the CH3 group to other alkyl group (ethyl, propyl, iso-propyl) led to a
reduction gradually in potency. In particular, replacement of CH3 group at position-R’’
with relatively big steric group including 2-hydroxy-2-methylethyl group and benzyl
group resulted in a completely loss of activity. Taken together, these results suggested
that the nature and size of R’’ group plays a vital role in determining the tyrosinase
inhibitory activity, and in the present investigation, the CH3 group is most optimal.
2.2.1.2. The inhibitory effect of biphenyl-type thiosemicarbazone compounds
For 2-(1-(4'-substituted-[1,1'-biphenyl]-4-yl)ethylidene)hydrazine-1-carbothio-
amide compounds, as shown in Fig. 4.B, structural influence factors on the inhibitory
activity are related to R’, R” and middle-phenyl.
(i) The inhibitory effect of R’: In this work, three series of biphenyl
thiosemicarbazone compounds bearing the representative R” that inculded alkxoy,
acyloxy and alyoxycarbonylmethyl substituents were investigated as the tyrosinase
inhibitors. As demonstrated in Table 1, compounds 8a-c give acceptable tyrosinase
inhibitory activities with the IC50 values of 1.513 µM, 1.992 µM and 2.446 µM,
respectively, while 8d-e have no activity even at the concentration of 50 µM. The
results revealed that the nature and size of R’ is crucial for maintaining the inhibitory

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activity, which was further verified by the data from compounds 8f-g. Interestingly,
compounds 9a-d exhibit potent tyrosinase inhibitory activities with the range of IC50
values from 0.278 to 0.562 µM. The result suggested that the introduction of
–CH2-part between alkoxycarbonyl and biphenyl moiety can be contributed to
tyrosinase inhibitory activity. Of note, compound 9e showed lower activity than 9a-d,
indicating that the hydrogen atom is unfavorable substituent for alkyl 2-(4'-(1-(2-
carbamothioylhydrazono)ethyl)-[1,1'-biphenyl]-4-yl)acetates. This result further
illustrated the hydrophobicity of the narrow “gap” in tyrosinase.
(ii) The inhibitory effect of R’’: As exemplified by 3h (R’’ = H) and 3i (R’’ = CH3),
we found that the latter (IC50 = 0.384 µM) would perform higher activity than the
former (IC50 = 2.781 µM). This conclusion is consistent with the above investigation
that obtained from the comparison of inhibitory activity of 4-alkyl- phenylalkan-1-one
with that of alkylbenzaldehyde thiosemicarbazone compounds.
(iii) The inhibitory effect of middle-phenyl moiety: By comparing the inhibitory
activity
of
2-(1-(4'-acyloxymethyl-[1,1'-biphenyl]-
4-yl)ethylidene)hydrazine-1-carbothioamide compounds (for 9a-c: the range of IC50
values from 0.278 to 0.485 µM) with that of the corresponding
1-(1-(4-acyloxymethylphenyl)ethylidene)thiosemicarbazide compounds (for 5a-c, the
range of IC50 values from 0.325 to 0.458 µM), we could concluded that the
introduction of middle-phenyl moiety has no obvious affect on the performance of the
inhibitors.
2.3. Inhibition mechanism of selected compounds 3d and 6e on mushroom
tyrosinase
The inhibition mechanism of compounds 3d and 6e on mushroom tyrosinase for the
oxidation of L-DOPA was first determined. Fig. 5 showed the relationship between
enzyme activity and concentration in the presence of different concentrations of
compounds 3d (A) and 6e (B). The results demonstrated that the plots gave a family
of straight lines, which all passed through the origin. Increasing the inhibitor
concentration resulted in the decrease in the slope of the line, thus revealing that
compounds 3d and 6e were reversible inhibitor.

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Fig. 5. The inhibitory effects of 3d (A) and 6e (B) on mushroom tyrosinase for the catalysis of
L-DOPA. The concentrations of compound 3d for curves 1-4 were 0.0, 0.1, 0.3 and 0.5 µM,
respectively. The concentrations of compound 6e for curves 1-4 were 0.0, 0.5, 1.0 and 2.0 µM,
respectively.
2.4. Inhibitory types of selected compounds 3d and 6e on mushroom tyrosinase
Fig. 6 Lineweaver–Burk plots for inhibition of selected compounds 3d and 6e on mushroom
tyrosinase for the catalysis of L-DOPA. Concentration of 3d for curves 1–3 were 0.5, 0.3 and 0.1
µM, respectively; 6e for curves 1–3 were 2.0, 1.0 and 0.5 µM, respectively.
To further insight into the inhibition mechanism, the inhibitory types of the
selected compounds 3d and 6e on mushroom tyrosinase for the oxidation of L-DOPA
were determined by the Line Weaver–Burk double reciprocal plots. Fig. 6 showed the
double-reciprocal plots of the enzyme inhibited by compound 3d (A). The results
displayed that the plots of 1/V versus 1/[S] gave three straight lines with different
slopes, but they intersected one another at the ordinate. The values of Vmax remained
the same and the values of Km increased with increasing concentrations of the

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inhibitor, which indicated that compound 3d was a competitive inhibitor of tyrosinase.
Identical research was carried out on 6e (B), and the same conclusion that 6e was the
competitive inhibitor on tyrosinase was drawn from (Fig. 6). These results revealed
that 3d and 6e could only bind with the free enzyme.
2.5. Toxicity of compounds 3d, 4e, 6a and 9a
Fig. 7 Results of cytotoxicity 3d, 4e, 6a and 9a to 293T cell line
In order to verify the safety of these thiosemicarbazone compounds, four
compounds 3d, 4e, 6a and 9a were selected to test their cytotoxicities in 293T cell
line. As demonstrated in Fig. 7, the results showed that these compounds had not an
obvious influence on the proliferation and the survival rate of 293T cells at the
concentrations of 0, 31.25, 62.5, 125, 250, 500, 1000 µmol/L.
Since compounds 3d, 4e, 6a and 9a were randomly selected and they all did not
perform any toxicity to 293T cell line even at a high concentration (1000 µmol/L), we
could conclude that these compounds involved in the study might present lower
cytotoxicities on 293T cell line. In combination with their effective inhibitory effects
on tyrosinase, therefore, these compounds should have broad application potential in
food industry, agriculture and cosmetics for the treatment of tyrosinase-related
disorders.
3. Conclusion
In summary, we developed several new series of substituted-benzaldehyde and

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substituted-phenylalkan-1-one and their biphenyl-type thiosemicarbazone analogues
bearing diverse R’ and R” part as tyrosinase inhibitors. The results showed that 22
compounds performed remarkable inhibitory activities with IC50 value of lower than
1.0 µM. The analysis of SARs indicated that: (1) the nature, length and space volume
of R’ played an important role in determining the inhibitory activity, and in the
present investigation, the proper length and relatively linear, oxygen atom-involved
and more hydrophobic group is optimal, (2) the nature and size of R’’ group
significantly influenced the tyrosinase inhibitory potency, and in this work, the CH3
group is the most favorable group, and (3) the introduction of middle-phenyl moiety
has no obvious affect on the performance of the inhibitors, while further introduction
of –CH2-part between alkoxycarbonyl and biphenyl moiety can be contributed to
tyrosinase inhibitory activity. Moreover, the inhibition mechanism and inhibition
kinetics study revealed that compound 3d and 6e was reversible and non-competitive
inhibitor. The evaluation of the cytotoxicity revealed that randomly selected
compounds 3d, 4e, 6a and 9a did not display toxicity to 293T cells even at a high
concentration of 1000 µmol/L. Taken together, these results suggested that such type
of thiosemicarbazone compounds could serve as a class of promising candidates for
the treatment of tyrosinase-related disorders. Inspired by these interesting data, further
investigations of these compounds using human tyrosinase and human melanoma cell
line are underway in our laboratory, and the research results will be reported in due
course.
4. Experimental
4.1. General
Melting points were determined on a WRS-1B digital instrument without
correction. NMR spectra were recorded on a Varian Mercury-Plus 300 spectrometer
or 400 spectrometer in DMSO-d6. All chemical shifts (δ) were quoted in parts per
million and coupling constants (J) were given in Hertz. Mass spectra were obtained
from VG ZAB-HS, LCMS-2010A or LCQ DECA XP spectrometer. Elemental
analyses were performed on a Vario EL instrument and were within ±0.4% of the
theoretical values. All commercially available reagents and solvents were used
without further purification. Mushroom tyrosinase (specific activity of the enzyme is
6680 U/mg) and L-DOPA (L-3,4-dhydroxyphenylalanine) were purchased from

ACCEPTED MANUSCRIPT
Sigma Chemical Co.
4.2. Procedure for the synthesis of target thiosemicarbazone compounds
4.2.1. Procedure for the synthesis of target thiosemicarbazone compounds 3a-j
Thiosemicarbazone compounds 3a-j were synthesized by condensation reaction
of the corresponding commercial available aldehyde or ketone compounds and
thiosemicarbazide (Scheme 1) using the previously reported method [15,16].
4.2.1.1. 1-(4-(iso-Propyl)benzylidene)thiosemicarbazide (3a)
Solid product, yield 88%, m.p. 144-147 oC. 1H NMR (300 MHz, DMSO-d6) δ:
11.36 (s, 1H), 8.16 (s, 1H), 8.00 (s, 1H), 7.91 (s, 1H), 7.68 (d, J = 7.6 Hz, 2H), 7.24 (d,
J = 7.5 Hz, 2H), 2.95-2.81 (m, 1H), 1.18 (d, J = 6.6 Hz, 6H). 13C NMR (75 MHz,
DMSO-d6) δ: 178.4, 151.0, 142.9, 132.5, 128.0, 127.3, 34.2, 24.5. ESI-MS (m/z )
222.1 [M+H]+. It was identified with the reported data [21].
4.2.1.2. 1-(1-(3’-Methylphenyl)ethylidene)thiosemicarbazide (3b)
1
Solid product, yield 78%, 189-191oC. H NMR (400 MHz, DMSO-d6) δ: 10.39
(s, 1H), 8.31 (s, 1H), 7.94 (s, 1H), 7.79 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.29 (t, J =
8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 2.35 (s, 3H), 2.31 (s, 3H). 13C NMR (75 MHz,
DMSO-d6) δ: 178.8, 148.0, 137.5, 137.4, 129.8, 128.1, 127.0, 123.8, 21.0, 14.0.
ESI-MS m/z = 208.1 [M+H]+. It was identified with the reported data [22].
4.2.1.3. 1-(1-(3’,4’-Dimethylphenyl)ethylidene)thiosemicarbazide (3c)
1
Solid product, yield 82%, m.p. 181-183oC. H NMR (400 MHz, DMSO-d6) δ:
10.13 (s, 1H), 8.26 (s, 1H), 7.86 (s, 1H), 7.74 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 6.92 (d,
J = 8.0 Hz, 1H), 2.26 (s, 6H), 2.23 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 178.7,
148.1, 137.6, 136.1, 135.1, 129.3, 127.4, 124.1, 19.3, 19.2, 13.9. ESI-MS m/z = 222.1
[M+H]+.
4.2.1.4. 1-(1-(4-Butylphenyl)ethylidene)thiosemicarbazide (3d)
1
Solid product, yield 82%, m.p. 109-110oC. H NMR (300 MHz, DMSO-d6) δ:
10.15 (s, 1H), 8.23 (s, 1H), 7.86 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.1 Hz,
2H), 2.58 (t, J = 7.5 Hz, 2H), 2.26 (s, 3H), 1.62–1.47 (m, 2H), 1.29 (dq, J = 14.6, 7.2
Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 178.7, 147.9,

ACCEPTED MANUSCRIPT
143.6, 135.1, 128.1, 126.5, 34.5, 32.9, 21.7, 13.9, 13.7. ESI-MS m/z = 250.2 [M+H]+.
Anal. Calcd for C13H19N3S: C, 62.61; H, 7.68; N, 16.85; found: C, 62.75; H, 7.75; N,
16.94.
4.2.1.5. 1-(1-(4-tert-Butylphenyl)-2-hydroxy-2-methylpropylidene)thiosemicarbazide
(3e)
Solid product, yield 63%, m.p.215-216 oC. 1H NMR (300 MHz, DMSO-d6) δ:
8.48 (s, 1H), 8.13 (s, 1H), 7.80 (s, 1H), 7.55 (d, J = 7.7 Hz, 2H), 7.16 (d, J = 7.7 Hz,
2H), 5.13 (s, 1H), 1.32 (s, 9H), 1.27 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 178.6,
158.8, 152.3, 128.9, 128.4, 126.6, 73.1, 35.4, 31.8, 29.3.
4.2.1.6. 1-(1-(Naphthalen-8-yl)ethylidene)thiosemicarbazide (3f)
Solid product, yield 76%, m.p. 226 oC (dec.). 1H NMR (400 MHz, DMSO) δ:
10.42 (s, 1H), 8.37-8.35 (m, 2H), 8.29 (d, J = 12.0 Hz, 1H), 8.00-7.98 (m, 1H),
13
7.39-7.91 (m, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.55-7.52 (m, 2H), 2.43 (s, 3H). C
NMR (75 MHz, DMSO-d6) δ: 178.9, 147.6, 135.0, 133.2, 132.7, 128.5, 127.5, 127.4,
126.7, 126.5, 126.3, 124.1, 13.8. ESI-MS m/z = 244.1 [M+H]+.
4.2.1.7. 1-(1-(1,2,3,4-Tetrahydronaphthalen-5-yl)ethylidene)thiosemicarbazide (3g)
1
Solid product, yield 73%, m.p. 126-128oC. H NMR (300 MHz, DMSO-d6) δ:
10.15 (s, 1H), 8.26 (s, 1H), 7.88 (s, 1H), 7.63-7.60 (m, 2H), 7.03 (d, J = 7.8Hz, 1H),
13
2.72 (J = 8.1Hz, 4H), 2.26 (s, 3H), 1.73(s, 4H). C NMR (75 MHz, DMSO-d6) δ:
178.7, 148.1, 138.0, 136.4, 134.8, 128.7, 127.0, 123.7, 28.6, 22.6, 13.9. ESI-MS m/z =
274.1 [M+H]+.
4.2.1.8. 4.1. 2-([1,1'-biphenyl]-4-ylmethylene)hydrazine-1-carbothioamide (3h)
1
Solid product, yield 85%, m.p. 202-203 oC. H NMR (300 MHz, DMSO-d6) δ：
10.24 (s, 1H), 8.29 (s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.97 (s, 1H), 7.68 (dd, J = 13.5,
5.1 Hz, 4H), 7.46 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 2.32 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) δ：178.9, 147.4, 140.7, 139.4, 136.7, 129.0, 127.7, 127.2, 126.6,
126.4, 13.9. ESI-MS m/z = 256.1 [M+H]+. It was identified with the reported data
[23].

ACCEPTED MANUSCRIPT
4.2.1.9. 2-(1-([1,1’-Biphenyl]-4-yl)ethylidene)hydrazine-1-carbothioamide (3i)
Solid product, yield 83%, m.p. 204 oC (dec.). 1H NMR (300 MHz, DMSO-d6) δ:
10.24 (s, 1H), 8.29 (s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.97 (s, 1H), 7.68 (dd, J = 13.5,
5.1 Hz, 4H), 7.46 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 2.32 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) δ: 178.9, 147.4, 140.7, 139.4, 136.7, 129.0, 127.7, 127.2, 126.6,
126.4, 13.9. ESI-MS m/z = 270.2 [M+H]+.
4.2.1.10. 1-(4-Styrylbenzylidene)thiosemicarbazide (3j)
1
Solid product, yield 65%, m.p. 264-266 oC. H NMR (300 MHz, DMSO-d6) δ:
11.43 (s, 1H), 8.20 (s, 1H), 8.02 (s, 2H), 7.78 (d, J = 8.3 Hz, 2H), 7.61 (dd, J = 7.8,
4.2 Hz, 4H), 7.43–7.20 (m, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 177.8, 141.9,
138.4, 136.9, 133.4, 129.4, 128.7, 128.4, 127.8, 127.7, 126.7, 126.6. ESI-MS m/z =
282.1 [M+H]+. It was identified with the reported data [24].
4.2.2. Procedure for the synthesis of target thiosemicarbazone compounds 4a-p
The corresponding 4-alkylphenylalkan-1-one intermediates 4a’-p’ were prepared
by Friedel-Crafts reaction of the commercial available alkylbenzene and the
corresponding acyl chloride, intermediate 4a’-p’ reacted with thiosemicarbazide to
offer 4a-p (Scheme 1) using the method mentioned above.
4.2.2.1. 1-(1-(4-Methylphenyl)butylidene)thiosemicarbazide (4a)
1
Solid product, yield 79%, m.p. 118-119 oC. H NMR (300 MHz, DMSO-d6) δ:
10.32 (s, 1H), 8.22 (s, 1H), 7.84 (s, 1H), 7.76 (d, J = 7.5 Hz, 2H), 7.17 (t, J = 8.4 Hz,
2H), 2.81 (t, J = 7.1 Hz, 1H), 2.30 (s, 2H), 1.50-1.32 (m, 2H), 0.92 (t, J = 7.1 Hz, 3H).
13C NMR (75 MHz, DMSO-d6) δ: 179.3, 151.3, 139.3, 134.7, 129.6, 127.3, 28.4, 21.7,
20.5, 14.6. ESI-MS m/z =236.4 [M+H]+.
4.2.2.2. 1-(1-(4-Methylphenyl)-2-methylpropylidene)thiosemicarbazide (4b)
1
Solid product, yield 81%, m.p. 102-103 oC. H NMR (300 MHz, DMSO-d6) δ:
8.48 (s, 1H), 8.25 (s, 1H), 7.77 (s, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 8.0 Hz,
2H), 2.87–2.70 (m, 1H), 2.35 (s, 3H), 1.09-0.99 (m, 6H). 13C NMR (75 MHz,
DMSO-d6) δ: 178.3, 158.4, 139.5, 130.5, 130.1, 127.5, 36.0, 21.4, 20.3. ESI-MS m/z
= 236.4 [M+H]+.

ACCEPTED MANUSCRIPT
4.2.2.3. 1-(1-(4-Methylphenyl)-2-phenylethylidene)thiosemicarbazide (4c)
1
Solid product, yield 87%, m.p.157-158 oC. H NMR (300 MHz, DMSO-d6) δ:
10.42 (s, 1H), 8.33 (s, 1H), 8.00 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.25 (t, J = 7.3 Hz,
2H), 7.19–7.05 (m, 5H), 4.33 (s, 2H), 2.27 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ:
178.9, 148.0, 138.8, 136.4, 134.0, 128.9, 128.6, 128.0, 127.0, 126.3, 31.5,
20.8.ESI-MS m/z =284.1 [M+H]+. Anal. Calcd for C16H17N3S: C, 67.81; H, 6.05; N,
14.83; found: C, 68.10; H, 6.08; N, 14.72.
4.2.2.4. 1-(1-(4-iso-Propylphenyl)-2-methylpropylidene)thiosemicarbazide (4d)
Solid product, yield 81%, m.p.107-108 oC. 1H NMR (300 MHz, DMSO-d6) δ:
8.48 (s, 1H), 8.27 (s, 1H), 7.77 (s, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.2 Hz,
2H), 3.03–2.88 (m, 1H), 2.87–2.73 (m, 1H), 1.23 (d, J = 6.9 Hz, 6H), 1.05 (d, J = 6.9
13
Hz, 6H). C NMR (75 MHz, DMSO-d6) δ: 178.3, 158.3, 150.1, 130.6, 127.9, 127.6,
36.0, 33.7, 24.1, 20.4. ESI-MS m/z = 264.2 [M+H]+. Anal. Calcd for C14H21N3S: C,
63.84; H, 8.04; N, 15.95; found: C, 63.91; H, 8.02; N, 15.91.
4.2.2.5. 1-(1-(4-(2-Methylpropyl)phenyl)ethylidene)thiosemicarbazide (4e)
1
Solid product, yield 81%, m.p. 107-108 oC. H NMR (300 MHz, DMSO-d6) δ:
10.14 (s, 1H), 8.20 (s, 1H), 7.84 (s, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.3 Hz,
2H), 2.67–2.52 (m, 1H), 2.26 (s, 3H).1.61-1.47 (m, 2H), 1.16 (d, J = 6.9 Hz, 3H), 0.74
13
(t, J = 7.3 Hz, 3H). C NMR (75 MHz, DMSO-d6) δ: 178.7, 148.4, 148.1, 135.3,
126.8, 126.6, 40.6, 30.4, 21.6, 13.9, 12.0. ESI-MS m/z = 250.2 [M+H]+. Anal. Calcd
for C13H19N3S: C, 62.61; H, 7.68; N, 16.85; found: C, 62.79; H, 7.72; N, 16.92.
4.2.2.6. 1-(1-(4-tert-Butylphenyl)ethylidene)thiosemicarbazide (4f)
1
Solid product, yield 74%, m.p. 166-167 oC. H NMR (300 MHz, DMSO-d6) δ:
10.17 (s, 1H), 8.25 (s, 1H), 7.84 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.9 Hz,
2H), 2.27 (s, 3H), 1.25 (s, 9H). 13C NMR (75 MHz, DMSO-d6) δ: 178.8, 151.8, 147.9,
134.9, 126.3, 124.9, 34.3, 30.9, 14.0. ESI-MS m/z = 250.2 [M+H]+. Anal. Calcd for
C13H19N3S: C, 62.61; H, 7.68; N, 16.85; found: C, 62.88; H, 7.79; N, 16.97.
4.2.2.7. 1-(1-(4-Butylphenyl)propylidene)thiosemicarbazide (4g)
1
Solid product, yield 78%, m.p. 88-89 oC. H NMR (300 MHz, DMSO-d6) δ:
10.26 (s, 1H), 8.23 (s, 1H), 7.85 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.2 Hz,

ACCEPTED MANUSCRIPT
2H), 2.84 (q, J = 7.4 Hz, 2H), 2.57 (t, J = 7.5 Hz, 2H), 1.61-1.47 (m, 2H), 1.29 (dq, J
= 14.2, 7.2 Hz, 2H), 1.00 (t, J = 7.5 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (75
MHz, DMSO-d6) δ: 178.7, 151.8, 143.6, 133.8, 128.3, 126.8, 34.5, 32.9, 21.8, 19.2,
13.7, 11.0. ESI-MS m/z = 264.3 [M+H]+. Anal. Calcd for C14H21N3S: C, 63.84; H,
8.04; N, 15.95; found: C, 63.88; H, 8.03; N, 15.97.
4.2.2.8. 1-(1-(4-Butylphenyl)butylidene)thiosemicarbazide (4h)
1
Solid product, yield 71%, m.p. 74-75 oC. H NMR (300 MHz, DMSO-d6) δ:
10.32 (s, 1H), 8.21 (s, 1H), 7.83 (s, 1H), 7.77 (d, J = 8.1 Hz, 2H), 7.16 (d, J = 7.9 Hz,
2H), 2.81 (t, J = 7.5 Hz, 2H), 2.56 (t, J = 7.5 Hz, 2H), 1.61-1.47 (m, 2H), 1.48-1.34
13
(m, 2H), 1.36-1.20 (m, 2H), 0.98-0.83 (m, 6H). C NMR (75 MHz, DMSO-d6) δ:
178.7, 150.6, 143.5, 134.2, 128.2, 126.6, 34.5, 33.0, 27.5, 21.7, 19.6, 13.7, 13.4.
ESI-MS m/z = 278.3 [M+H]+. Anal. Calcd for C15H23N3S: C, 64.94; H, 8.36; N, 15.15;
found: C, 65.11; H, 8.37; N, 15.16.
4.2.2.9. 1-(1-(4-Pentylphenyl)ethylidene)thiosemicarbazide (4i)
1
Solid product, yield 78%, m.p. 123-124 oC. H NMR (300 MHz, DMSO-d6) δ:
10.22 (s, 1H), 8.30 (s, 1H), 7.90 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz,
2H), 2.55 (t, J = 7.5 Hz, 2H), 2.27 (s, 3H), 1.62–1.44 (m, 2H), 1.36–1.13 (m, 4H),
0.83 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 179.3, 148.3, 144.1, 135.6,
128.6, 127.0, 35.3, 31.3, 31.0, 22.5, 14.4, 14.4. ESI-MS m/z = 264.3 [M+H]+. Anal.
Calcd for C14H21N3S: C, 63.84; H, 8.04; N, 15.95; found: C, 63.78; H, 8.03; N, 15.87.
4.2.2.10. 1-(1-(4-Hexylphenyl)ethylidene)thiosemicarbazide (4j)
Solid product, yield 70%, m.p. 115-116 oC. 1H NMR (300 MHz, DMSO-d6) δ:
10.21 (s, 1H), 8.29 (s, 1H), 7.89 (s, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.2 Hz,
2H), 2.57 (t, J = 8.0 Hz, 2H), 2.30 (d, J = 7.3 Hz, 3H), 1.63-1.47 (m, 2H), 1.36-1.14 m,
6H), 0.84 (t, J = 6.2 Hz, 4H).13C NMR (75 MHz, DMSO-d6) δ: 179.3, 148.3, 144.1,
135.6, 128.6, 127.0, 35.4, 31.6, 31.3, 28.8, 22.6, 14.4. ESI-MS m/z = 278.3 [M+H]+.
Anal. Calcd for C15H23N3S: C, 64.94; H, 8.36; N, 15.15; found: C, 64.91; H, 8.34; N,
15.03.
4.2.2.11. 1-(1-(4-Cyclohexylphenyl)ethylidene)thiosemicarbazide (4k)
1
Solid product, yield 70%, m.p. 130-131 oC. H NMR (300 MHz, DMSO-d6) δ:

ACCEPTED MANUSCRIPT
10.21 (s, 1H), 8.28 (s, 1H), 7.89 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz,
2H), 2.58-2.44 (m, 1H), 2.28 (s, 3H), 1.88-1.68 (m, 4H), 1.48-1.13 (m, 6H). 13C NMR
(75 MHz, DMSO-d6) δ: 179.3, 148.3, 144.1, 135.6, 128.6, 127.0, 35.4, 31.6, 31.3,
28.8, 22.6, 14.4. ESI-MS m/z = 276.3 [M+H]+.
4.2.2.12. 1-(1-(4-Cyclohexylphenyl)propylidene)thiosemicarbazide (4l)
1
Solid product, yield 78%, m.p. 133-134 oC. H NMR (300 MHz, DMSO-d6) δ:
10.27 (s, 1H), 8.23 (s, 1H), 7.84 (s, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.3 Hz,
2H), 2.84 (q, J = 7.4 Hz, 2H), 2.58-2.42 (m, 2H), 1.76 (d, J = 9.2 Hz, 4H), 1.48-1.13
13
(m, 6H), 1.00 (t, J = 7.5 Hz, 3H). C NMR (75 MHz, DMSO-d6) δ: 178.8, 151.8,
148.7, 134.0, 126.9, 126.6, 43.6, 33.7, 26.3, 25.5, 19.2, 11.0.ESI-MS m/z = 290.3
[M+H]+. Anal. Calcd for C16H23N3S: C, 66.39; H, 8.01; N, 14.52; found: C, 66.68;
H, 8.11; N, 14.24.
4.2.2.13. 1-(1-(4-Cyclohexylphenyl)butylidene)thiosemicarbazide (4m)
Solid product, yield 75%, m.p. 121-122oC. 1H NMR (300 MHz, DMSO-d6) δ:
10.33 (s, 1H), 8.22 (s, 1H), 7.82 (s, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.3 Hz,
2H), 2.87-2.76 (m, 2H), 2.51-2.47 (m, 1H), 1.77 (d, J = 9.3 Hz, 4H), 1.49-1.31 (m,
6H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 178.7, 150.6, 148.6,
134.4, 126.7, 126.6, 43.5, 33.7, 27.6, 26.3, 25.5, 19.6, 13.7. ESI-MS m/z = 304.4
[M+H]+.
4.2.2.14. 1-(1-(4-Cyclohexylphenyl)-2-methylpropylidene)thiosemicarbazide (4n)
1
Solid product, yield 73%, m.p. 148-149 oC. H NMR (300 MHz, DMSO-d6) δ:
8.41 (s, 1H), 8.22 (s, 1H), 7.73 (s, 1H), 7.38 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz,
2H), 2.80-2.69 (m, 1H), 2.60-2.51 (m, 1H), 1.90-1.73 (m, 4H), 1.49-1.30 (m, 6H),
1.05 (d, J = 6.8 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ: 177.7, 157.9, 148.8, 130.1,
127.7, 127.1, 43.4, 35.5, 33.6, 26.3, 25.5, 19.7. ESI-MS m/z = 304.4 [M+H]+.
4.2.2.15. 1-(1-(4-Cyclohexylphenyl)-2-phenylethylidene)thiosemicarbazide (4o)
1
Solid product, yield 87%, m.p. 150-151 oC. H NMR (300 MHz, DMSO-d6) δ:
10.43 (s, 1H), 8.33 (s, 1H), 7.99 (s, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.26 (t, J = 7.3 Hz,
2H), 7.20-7.07 (m, 5H), 4.33 (s, 2H), 2.53-2.43 (m, 1H), 1.81-1.68 (m, 4H), 1.45-1.16
13
(m, 6H). C NMR (75 MHz, DMSO-d6) δ: 178.9, 148.8, 147.9, 136.4, 134.5, 128.6,

ACCEPTED MANUSCRIPT
128.4, 127.0, 126.6, 126.3, 43.4, 33.6, 31.6, 26.2, 25.5.ESI-MS m/z =352.4 [M+H]+.
Anal. Calcd for C21H25N3S: C, 71.75; H, 7.17; N, 11.95; found: C, 71.69; H, 7.25; N,
11.86.
4.2.2.16. 1-(1,2-Diphenylethylidene)thiosemicarbazide (4p)
Solid product, yield 78%, m.p.148-149 oC. 1H NMR (300 MHz, DMSO-d6) δ:
10.50 (s, 1H), 8.38 (s, 1H), 8.05 (s, 1H), 7.98-7.88 (m, 2H), 7.37-7.28 (m, 3H), 7.25 (t,
J = 7.2 Hz, 2H), 7.19-7.08 (m, 3H), 4.37 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ:
179.0, 147.8, 136.8, 136.4, 129.1, 128.6, 128.3, 128.0, 127.0, 126.3, 31.6. ESI-MS
m/z = 270.2 [M+H]+.
4.2.3. Procedure for the synthesis of target thiosemicarbazone compounds 5a-d,
6a-d and 7a-d
The acetylation of alkyl phenylacetates, alkyl 3-phenylpropanoate or alkyl
4-phenylbutanoates formed alkyl 4-acetylphenylacetates 5a’-d’, alkyl 3-(4-acetyl-
phenyl)propanoates 6a’-d’ or alkyl 4-(4-acetylphenyl)butanoates 7a’-c’, these
intermediates reacted with thiosemicarbazide offering thiosemicarbazone analogues
5a-d, 6a-d and 7a-c (Scheme 1).
4.2.3.1. Methyl 4-(1-thiosemicarbazidoethyl)phenylacetate (5a)
Solid product, yield 68%, m.p.157.2-157.8 oC. 1H NMR (300 MHz, DMSO-d6) δ:
10.24 (s, 1H), 8.34-8.29 (m, 1H), 7.94 (s, 1H), 7.87 (d, J = 8.1 Hz 2H), 7.26 (d, J =
13
8.1 Hz 2H), 3.74-3.72 (m, 2H), 3.62 (s, 3H), 2.29 (s, 3H). C NMR (75 MHz,
DMSO-d6) δ: 178.9, 171.6, 147.5, 136.3, 135.4, 129.2, 126.6, 51.7, 13.9. ESI-MS m/z
= 266.1 [M+H]+.
4.2.3.2. Ethyl 4-(1-thiosemicarbazidoethyl)phenylacetate (5b)
Solid product, yield 71%, m.p.145.2-146.5 oC. 1H NMR (300 MHz, DMSO-d6) δ:
10.22 (s, 1H), 8.35-8.30 (m, 1H), 7.93 (s, 1H), 7.86 (d, J = 8.1 Hz 2H), 7.28 (d, J =
8.1 Hz 2H), 3.73-3.72 (m, 2H), 4.12 (q, J = 7.2 Hz 2H), 2.30 (s, 3H), 1.21 (t, J = 7.5
Hz, 3H ). 13C NMR (75 MHz, DMSO-d6) δ: 179.0, 171.4, 147.4, 136.6, 135.8, 129.4,
126.9, 60.8, 14.0. ESI-MS m/z = 280.1 [M+H]+.
4.2.3.3. Propyl 4-(1-thiosemicarbazidoethyl)phenylacetate (5c)

ACCEPTED MANUSCRIPT
Solid product, yield 78%, m.p.151.4-152.7 oC. 1H NMR (300 MHz, DMSO-d6) δ:
10.25 (s, 1H), 8.34-8.30 (m, 1H), 7.94 (s, 1H), 7.85 (d, J = 8.1 Hz 2H), 7.27 (d, J =
8.1 Hz 2H), 4.12 (q, J = 7.2 Hz 2H), 3.74-3.71 (m, 2H), 2.31 (s, 3H), 1.75-1.73 (m,
2H), 1.00 (t, J = 7.5 Hz, 3H ). 13C NMR (75 MHz, DMSO-d6) δ: 178.9, 171.3, 147.3,
136.5, 135.6, 129.3, 126.5, 65.7, 22.0, 14.2, 11.2. ESI-MS m/z = 294.1 [M+H]+.
4.2.3.4. Buyl 4-(1-thiosemicarbazidoethyl)phenylacetate (5d)
Solid product, yield 73%, m.p.142.5-143.3 oC. 1H NMR (300 MHz, DMSO-d6) δ:
10.25 (s, 1H), 8.35-8.31 (m, 1H), 7.95 (s, 1H), 7.84 (d, J = 8.1 Hz 2H), 7.28 (d, J =
8.1 Hz 2H), 4.12 (q, J = 7.2 Hz 2H), 3.74-3.71 (m, 2H), 2.30 (s, 3H), 1.55-1.58 (m,
2H), 1.45-1.40 (m, 2H), 0.91 (t, J = 7.5 Hz, 3H ). 13C NMR (75 MHz, DMSO-d6) δ:
179.2, 171.0, 147.22, 136.4, 135.8, 129.5, 126.3, 65.0, 31.0, 19.0, 14.7, 13.2. ESI-MS
m/z = 308.1 [M+H]+.
4.2.3.5. Methyl 3-(4-(1-thiosemicarbazidoethyl)phenyl)propanoate (6a)
Solid product, yield 68%, m.p.177.5-178.6 oC. 1H NMR (400 MHz, DMSO-d6) δ:
8.81 (s, 1H), 7.65 (d, J = 7.0 Hz, 2H), 7.37 (s, 1H), 7.25 (d, J = 8.0, Hz 2H), 6.49 (s,
1H), 3.69 (s, 3H), 3.00 (t, J = 8.0 Hz, 2H), 2.67 (t, J = 8.0 Hz, 2H), 2.30 (s, 3H). 13C
NMR (75 MHz, DMSO-d6) δ: 173.1, 148.0, 142.8, 135.3, 129.8, 128.6, 126.6, 51.7,
35.3, 30.6, 13.5. ESI-MS m/z = 280.1 [M+H]+.
4.2.3.6. Ethyl 3-(4-(1-thiosemicarbazidoethyl)phenyl)propanoate (6b)
Solid product, yield 72%, m.p.139.3-149.5 oC. 1H NMR (400 MHz, DMSO-d6) δ:
8.81 (s, 1H), 7.64 (d, J = 8.0, Hz, 2H), 7.38 (s, 1H), 7.20 (d, J = 8.0 Hz, 2H), 6.48 (s,
1H), 4.14 (q, J = 8.0 Hz, 2H), 3.00 (t, J = 8.0 Hz, 2H), 2.65 (t, J = 8.0 Hz, 2H), 2.30 (s,
3H), 1.26 (t, J = 8.0 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ: 172.6, 148.0, 142.9,
135.2, 128.6, 126.7, 126.6, 60.5, 35.6, 30.6, 14.2, 13.5. ESI-MS m/z = 294.1 [M+H]+.
4.2.3.7. Propyl 3-(4-(1-thiosemicarbazidoethyl)phenyl)propanoate (6c)
Solid product, yield 81%, m.p.145.4-146.5 oC. 1H NMR (400 MHz, DMSO-d6) δ:
8.88 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.38 (s, 1H), 7.25 (d, J = 8.0 Hz, 2H), 6.55 (s,
1H), 4.04 (q, J = 8.0 Hz, 2H), 3.00 (t, J = 8.0 Hz, 2H), 2.66 (t, J = 8.0 Hz, 2H), 2.30 (s,
13
3H), 1.69-1.58 (m, 2H), 0.92 (t, J = 8.0 Hz, 3H). C NMR (75 MHz, DMSO-d6) δ:
179.3, 147.8, 143.0, 134.7, 128.6, 126.6, 125.5, 66.2, 35.5, 30.7, 22.0, 13.4, 10.4.

ACCEPTED MANUSCRIPT
ESI-MS m/z = 308.1 [M+H]+.
4.2.3.8. 3-(4-(1-Thiosemicarbazidoethyl)phenyl)propanoic acid (6d)
The hydrolysis of 6a produced solid product 6d, yield 65%, m.p.196.9-197.4 oC.
1H NMR (400 MHz, DMSO-d6) δ: 12.13 (s, 1H), 10.18 (s, 1H), 8.25 (s, 1H), 7.90 (s,
1H), 7.83 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 2.84 (t, J = 8.0 Hz, 2H), 2.5 (t,
J = 8.0 Hz, 2H), 2.28 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 178.8, 173.7, 147.9,
142.2, 135.5, 128.3, 126.6, 34.6, 30.2, 13.9. ESI-MS m/z = 264.1 [M-H]-.
4.2.3.9. Methyl 4-(4-(1-thiosemicarbazidoethyl)phenyl)butanoate (7a)
1
Solid product, yield 77%, m.p.147.3-147.7 oC. H NMR (300MHz,DMSO-d6)
δ:
10.16 (s, 1H), 8.24 (s, 1H), 7.87(s, 1H), 7.80 (d, J = 9.0 Hz, 2H), 7.15(d, J = 9.0 Hz,
2H), 3.56 (s, 3H), 2.59 (t, J = 9.0 Hz, 2H), 2.25-2.31 (m, 5H), 1.85-1.76 (m, 2H). 13C
NMR (75MHz, DMSO-d6) δ: 178.8, 173.1, 147.8, 142.7, 135.4, 128.6, 126.6, 51.2,
34.2, 32.7, 26.0,13.9. ESI-MS m/z = 294.1 [M+H]+.
4.2.3.10. Ethyl 4-(4-(1-thiosemicarbazidoethyl)phenyl)butanoate (7b)
1
Solid product, yield 69%, m.p.130.2-130.6 oC. HNMR (300MHz,DMSO-d6)
δ:
10.14 (s, 1H), 8.22 (s, 1H), 7.86 (s, 1H), 7.80 (d, J = 9.0 Hz, 2H), 7.15 (d, J = 9.0 Hz,
2H), 4.01 (t, J = 6.0 Hz, 2H), 2.59 (t, J = 6.0 Hz, 2 H), 2.26-2.24 (m,5H), 1.78-1.85
13
(m, 2H), 1.16 (t, J = 6.0 Hz, 3H). C NMR (75MHz, DMSO-d6) δ: 178.8, 172.6,
147.8, 142.7, 135.4, 128.2, 126.6, 59.7, 33.9, 32.8, 26.1, 14.1, 13.9. ESI-MS m/z =
308.1 [M+H]+.
4.2.3.11. Butyl 4-(4-(1-thiosemicarbazidoethyl)phenyl)butanoate (7c)
o
1
Solid product, yield 73%, m.p.116.9-117.8 C. HNMR (300MHz,DMSO-d6)
:10.17 (s, 1H), 8.24 (s, 1H), 7.88 (s, 1H), 7.81 (d, J = 9.0 Hz, 2H), 7.15 (d, J = 9.0 Hz,
δ
2H), 3.99 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 9.0 Hz, 2 H), 2.31-2.26 (m, 5H), 1.86-1.79
13
(m, 2H), 1.56-1.51 (m, 2H), 1.35-1.27 (m, 2H), 0.88 (t, J = 6.0 Hz, 3H). C NMR
(75MHz, DMSO-d6) δ: 178.8, 172.6, 147.8, 142.7, 135.4, 128.2, 126.6, 63.4, 34.0,
32.9, 30.2, 26.1, 18.6, 13.9,13.5. ESI-MS m/z = 336.2 [M+H]+.
4.2.4. Procedure for the synthesis of target thiosemicarbazone compounds 8a-j
Compounds 8a-j were obtained as the method shown in Scheme 1. [1,1'-Bi-

ACCEPTED MANUSCRIPT
phenyl]-4-ol reacted with acyl chloride forming [1,1'-biphenyl]-4-yl alkanoates,
which reacted with acetic chloride to give 4'-acetyl-[1,1'-biphenyl]-4-yl alkanoates
8a’-e’. 8a’-e’ Reacted with thiosemicarbazide to produce compounds 8a-e.
[1,1'-Biphenyl]-4-ol reacted with alkyl bromide forming 4-alkoxy-[1,1'-biphenyl],
which reacted with acetic chloride to give 4-alkoxy-4'-acetyl-[1,1'-biphenyl], 8g’-j’.
8g’-j’ Reacted with thiosemicarbazide to produce the target compounds 8g-j. The
hydrolysis of 8a offered compound 8f.
4.2.4.1. 2-(1-(4’-Acetoxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbothio-
amide (8a)
o
1
Solid product, yield 76%, m.p. >245 C. H NMR (300 MHz, DMSO-d6) δ:
10.24 (s, 1H), 8.29 (s, 1H), 7.99 (s, 3H), 7.69 (d, J = 12.5 Hz, 4H), 7.21 (d, J = 7.4 Hz,
2H), 2.32 (s, 3H), 2.29 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 176.7, 167.2, 148.2,
145.2, 137.8, 135.0, 134.7, 125.7, 125.2, 124.5, 119.9, 18.8, 11.9. ESI-MS m/z =
328.1 [M+H]+.
4.2.4.2. 2-(1-(4’-iso-Butyroxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbo-
thioamide (8b)
Solid product, yield 64%, m.p. 245 oC (dec.). 1H NMR (300 MHz, DMSO-d6) δ:
10.25 (s, 1H), 8.30 (s, 1H), 8.01 (d, J = 8.2 Hz, 3H), 7.69 (dd, J = 20.8, 7.7 Hz, 4H),
13
7.19 (d, J = 7.5 Hz, 2H), 2.83 (s, 1H), 2.32 (s, 3H), 1.25 (s, 3H), 1.23 (s, 3H). C
NMR (75 MHz, DMSO-d6) δ: 178.8, 175.0, 150.3, 147.3, 139.8, 140.0, 136.7, 127.7,
127.2, 126.4 , 122.3, 33.3, 18.7, 13.7. ESI-MS m/z = 356.1 [M+H]+.
4.2.4.3. 2-(1-(4’-Pivaloyloxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbo-
thioamide (8c)
1
Solid product, yield 75%, m.p. 240oC (dec.). H NMR (300 MHz, DMSO-d6) δ:
10.25 (s, 1H), 8.31 (s, 1H), 8.01 (d, J = 8.5 Hz, 3H), 7.69 (dd, J = 21.1, 8.5 Hz, 4H),
7.17 (d, J = 8.5 Hz, 2H), 2.32 (s, 3H), 1.31 (s, 9H). 13C NMR（75 MHz, DMSO-d6）
δ: 178.8, 176.4, 1505, 147.3, 139,8, 136.9, 136.6, 127.7, 127.2, 126.4, 122.2, 26.7,
13.9. ESI-MS m/z = 370.2 [M+H]+.
4.2.4.4. 2-(1-(4’-Benzoyloxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbo-
thioamide (8d)

ACCEPTED MANUSCRIPT
Solid product, yield 77%, m.p. 243 oC (dec.). 1H NMR (300 MHz, DMSO-d6) δ:
10.26 (s, 1H), 8.31 (s, 1H), 8.14 (d, J = 7.2 Hz, 2H), 8.03 (d, J = 8.5 Hz, 3H), 7.82-
13
7.65 (m, 5H), 7.60 (t, J = 7.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H), 2.33 (s, 3H). C
NMR (75 MHz, DMSO-d6) δ: 178.8, 164.6, 150.3, 147.3, 139.8, 137.2, 136.7, 134.1,
129.8, 129.0, 128.8, 127.8, 127.2, 126.4, 122.5, 13.9. ESI-MS m/z = 390.1 [M+H]+.
4.2.4.5. 2-(1-(4’-(4-Methoxybenzoyloxy)-[1,1’-biphenyl]-4-yl)ethylidene)-
hydrazine-1-carbothioamide (8e)
Solid product, yield 62%, m.p. 238 oC (dec.). 1H NMR (300 MHz, DMSO-d6) δ:
10.25 (s, 1H), 8.30 (s, 1H), 8.05 (dd, J = 18.2, 7.0 Hz, 6H), 7.88-7.64 (m, 4H), 7.35 (d,
13
J = 7.1 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 3.86 (s, 3H), 2.33 (s, 3H). CNMR (75
MHz, DMSO-d6) δ: 181.0, 165.8, 148.3, 143.1，137.1, 131.3, 129.7, 129.5, 128.0,
122.1, 114.2, 55.8, 17.3. ESI-MS m/z = 420.1 [M+H]+.
4.2.4.6.
2-(1-(4’-Hydroxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbothio-
amide (8f)
Solid product, yield 75%, m.p. >245oC. 1H NMR (300 MHz, DMSO-d6) δ: 10.20
(s, 1H), 9.62 (s, 1H), 8.26 (s, 1H), 7.93 (s, 3H), 7.61-7.45 (m, 4H), 6.84 (d, J = 6.8 Hz,
2H), 2.30 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ：178.8, 157.3, 147.6, 140.7, 135.6,
130.0, 127.7, 127.0, 125.5, 115.8, 13.9. ESI-MS m/z = 286.1 [M+H]+.
4.2.4.7. 2-(1-(4’-Ethoxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbo-
thioamide (8g)
1
Solid product, yield 58%, m.p. 245 oC(dec.). H NMR (300MHz, DMSO-d6)
δ:
10.20 (s, 1H), 8.26 (s, 1H), 7.97-7.94 (m, 3H), 7.63-7.58 (m, 4H), 6.97 (d, J = 6.0 Hz,
2H), 4.04 (q, J = 6.0 Hz, 2H), 2.31 (s, 3H), 1.34 (t, J = 4.0 Hz, 3H). 13C NMR
(75MHz, DMSO-d6) δ: 178.8, 158.4, 147.5, 140.4, 135.9, 131.5, 127.7, 127.1, 125.7,
114.8, 63.1, 14.6, 13.9. ESI-MS m/z = 314.1 [M+H]+.
4.2.4.8. 2-(1-(4’-Propoxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbo-
thioamide (8h)
1
Solid product, yield 75%, m.p. 245 oC(dec.). HNMR (300MHz, DMSO-d6)
δ:
10.20 (s, 1H), 8.26 (s, 1H), 7.98-7.94 (m, 3H), 7.63-7.58 (m, 4H), 7.01-6.97 (m, 2H),

ACCEPTED MANUSCRIPT
3.96 (t, J = 6.0 Hz, 2H), 2.31 (s, 3H), 1.78-1.69 (m, 2H), 0.97 (t, J = 6.0 Hz, 3H). 13C
NMR (75MHz, DMSO-d6) δ: 178.8, 158.5, 147.5, 140.4, 135.9, 131.5, 127.7, 127.1,
125.7, 114.9, 69.0, 22.0, 13.9, 10.4. ESI-MS m/z = 328.1 [M+H]+.
4.2.4.9.
2-(1-(4’-Butoxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbothio-
amide (8i)
Solid product, yield 63%, m.p. 245 oC(dec.). H NMR (300MHz, DMSO-d6)
1
δ:
10.20 (s, 1H), 8.25 (s, 1H,), 7.92-7.28 (m, 3H,), 7.63-7.58 (d, 4H,), 7.01-6.97 (m, 2H,),
4.02-3.95 (m, 2H,), 2.31 (s, 3H), 1.73-1.65 (m, 2H,) 1.48-1.39 (m, 2H), 0.92 (t, J =
13
6.0 Hz, 3H). C NMR (75MHz, DMSO-d6) δ: 178.8, 158.6, 147.5, 140.4, 135.8,
131.5, 127.7, 127.1, 125.7, 114.9, 67.2, 30.7, 18.7, 13.9, 13.7. ESI-MS m/z = 342.2
[M+H]+.
4.2.4.10.
thioamide (8j)
Solid product, yield 65%, m.p. 245 oC(dec.). H NMR(300MHz, DMSO-d6)
2-(1-(4’-Pentoxy-[1,1’-biphenyl]-4-yl)ethylidene)hydrazine-1-carbo-
1
δ:
10.20 (s,1H), 8.29 (s,1H), 7.98-7.94 (m, 3H), 7.62-7.57 (m, 4H), 7.00-6.96 (d, 2H),
3.97(t, t, J = 6.0 Hz, 2H), 2.31 (s, 3H), 1.74-1.86 (m, 2H), 1.42-1.29 (m, 4H), 0.88(t, J
13
= 6.0 Hz, 3H). C NMR (75MHz, DMSO-d6) δ: 178.8, 158.5, 147.5, 140.4, 135.8,
131.5, 127.7, 127.1, 125.7, 114.9, 67.5, 28.3, 27.7, 21.9, 13.90, 13.8.
4.2.5. Procedure for the synthesis of target thiosemicarbazone compounds 9a-e
Compounds 9a-e were prepared as the method for synthesizing 5a-d, 6a-d or
7a-c (Scheme 5.).
4.2.5.1.
2-(1-(4’-(Methoxycarbonylmethyl)-[1,1’-biphenyl]-4-yl)ethylidene)-
hydrazine-1-carbothioamide (9a)
1
Solid product, yield 72%, m.p.198.1-199.5 oC. H NMR (300MHz, DMSO-d6)
δ: 10.27 (s, 1H), 8.32 (s, 1H), 8.04-8.01 (m, 3H), 7.66 (dd, J = 8.4 Hz, 1.5Hz, 4H),
13
7.36 (d, J = 8.1 Hz, 2H), 3.74 (s, 2H), 3.63 (s, 3H), 2.34 (s, 3H). C NMR (75MHz,
DMSO-d6) δ: 178.8, 171.5, 147.4, 140.3, 137.9, 136.6,133.9, 130.0, 127.2, 126.6,
126.3, 51.7, 13.9. ESI-MS m/z = 342.1 [M+H]+.

ACCEPTED MANUSCRIPT
4.2.5.2.
2-(1-(4’-(Ethoxycarbonylmethyl)-[1,1’-biphenyl]-4-yl)ethylidene)-
hydrazine-1-carbothioamide (9b)
Solid product, yield 74%, m.p.189.2-189.9 oC. H NMR (300MHz, DMSO-d6)
1
δ:
10.22 (s,1H), 8.27 (s, 1H), 8.01-7.98 (m, 3H), 7.63 (d, J = 9.0 Hz, H), 7.33 (d, J = 4.0
13
Hz, 2H), 4.06 (q, J = 6.0 Hz, 2H), 3.70 (s, 2H), 1.19 (t, J = 9.0 Hz, 3H). C NMR
(75MHz, DMSO-d6) δ: 178.8, 171.1, 147.4, 140.3, 137.9, 136.6, 134.0, 130.0, 127.2,
126.6, 126.3, 60.3, 14.1,13.9. ESI-MS m/z = 356.1 [M+H]+.
4.2.5.3. 2-(1-(4’-(Propoxycarbonylmethyl)-[1,1’-biphenyl]-4-yl)ethylidene)-
hydrazine-1-carbothioamide (9c)
o
1
Solid product, yield 75%, m.p.180.2-180.7 C. H NMR (300MHz, DMSO-d6)
:10.22 (s, 1H), 8.28 (s, 1H), 8.01-7.98 (m, 3H), 7.63 (d, J = 9.0 Hz, 4H), 7.33 (d, J =
δ
9.0 Hz, 2H), 3.99 (t, J = 4.0 Hz, 2H), 3.71 (s, 2H), 2.31 (s, 3H), 1.61-1.54 (m, 2H),
0.86 (t, J = 9.0 Hz, 3H). 13C NMR (75MHz, DMSO-d6) δ: 178.8, 171.1, 147.4, 140.3,
137.9, 136.6, 134.0, 130.0, 127.2, 126.7, 126.2, 65.7, 21.5, 13.9, 10.2. ESI-MS m/z =
370.2 [M+H]+.
4.2.5.4.
2-(1-(4’-(Butoxycarbonylmethyl)-[1,1’-biphenyl]-4-yl)ethylidene)
hydrazine-1-carbothioamide (9d)
1
Solid product, yield 71%, m.p.167.2-168.7 oC. H NMR (300MHz, DMSO-d6)
δ:10.22 (s,1H), 8.28 (s,1H), 8.01-7.98 (m, 3H), 7.63 (d, J = 9.0 Hz, 4H), 7.32 (d, J =
9.0 Hz, 2H), 4.04 (q, J = 6.0 Hz, 2H), 3.70 (s,3H), 2.31(s, 3H), 1.59-1.49 (m,2H), 1.33
-1.26 (m,2H), 0.86 (t, J = 6.0 Hz, 3H). 13C NMR (75MHz, DMSO-d6) δ: 178.8, 171.1,
147.4, 140.3, 137.9, 136.6, 134.0, 130.0, 127.2, 126.5, 126.2, 64.0, 30.1, 18.5, 13.9,
13.5. ESI-MS m/z = 384.2 [M+H]+.
4.3. Assay of inhibitory activities of target compounds
The inhibition of target compounds on the diphenolase activitiy of mushroom
tyrosinase was investigated by our reported procedure [15, 16] with some slight
modifications. Briefly, all the synthesized compounds were screened for the
diphenolase inhibitory activity of tyrosinase using L-DOPA as substrate.
Thiosemicarbazide, kojic acid (used as a control) and all the thiosemicarbazone
compounds were dissolved in DMSO and their final concentration in DMSO was

ACCEPTED MANUSCRIPT
2.0%. Phosphate buffer, pH 6.8, was used to dilute the DMSO stock solution of test
compounds. Thirty units of tyrosinase (0.5 mg/mL) was first pre-incubated with the
samples, in 50 mM phosphate buffer, for 10 min at 25 oC, L-DOPA solution (0.5 mM)
was added to the mixture and the enzyme reaction was monitored by measuring the
change in absorbance at 475 nm of formation of DOPAchrome for 1 min. The
measurement was completed in triplicate for each concentration and averaged before
further calculation. IC50 value was determined by interpolation of the dose-response
curves and the results were presented in Table 1.
4.4. Assay of inhibition mechanism and inhibitory types of selected compounds 3d
and 6e on mushroom tyrosinase
The assay of inhibition mechanism and inhibitory types of selected compounds
3d and 6e on mushroom tyrosinase was finished as reported protocol [15, 16]. The
results were shown in Fig. 5 and Fig. 6.
4.5. Assay of cytotoxicity of selected compounds 3d, 4e, 6a and 9a to 293T cell line
The assay of cytotoxicity of selected compounds to 293T cell line was finished
as the generally reported protocol [25]. Briefly, the 293T cell line suspension was
prepared and 100 µL of the suspension was injected in each tube which was placed in
96-hole culture plate, and the culture plate was pre-cultured in the incubator for 24 h
o
(37 C, 5% CO2); 10 µL of different-concentration solution of 3d was added to each
cell line tube and the final concentration of 3d was respectively kept in 31.25, 62.5,
125, 250, 500, 1000 µmol/L; The culture plate was incubated for 24 h in the incubator,
then 10 µL of CCK-8 solution was added into each tube in 96-hole plate; After the
culture plate was incubated for 4 h in the incubator, the absorbance of the mixture in
each tube at 450 nm was determined using micro-plate reader.
The cell line tube to which compound 3d was not injected as reference and the
cell-proliferation number of this cell line was assumed “1”. At this time the cell
proliferation number for each cell line tube was determined as 1.012, 1.086, 1.086,
1.027, 1.119, 1.014. With the same method, we also determined the cytotoxicity of
compounds 4e, 6a and 9a to 293T cell line.
Acknowledgement: This work was supported by The Planed Project of Science and
Technology of Guangdong Province (2014B010108016).

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