Rational design and synthesis of 4-O-substituted phenylmethylenethiosemicarbazones as novel tyrosinase inhibitors

Article abstract of DOI:10.1248/cpb.58.752

In continuing our program aimed to search for tyrosinase inhibitors, a series of novel 4-O-substituted phenylmethylenethiosemicarbazones were rational designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were also evaluated. A fair number of compounds were found to have significant tyrosinase inhibitiory activity. Particularly, the IC₅₀ values of compounds 3a-g, 3j and 3s were of the same magnitude as tropolone, one of the best tyrosinase inhibitors known so far. Furthermore, the structure-activity relationships of these compounds were also investigated. All these data suggested that these molecules might be utilized for the development of new candidate for the treatment of dermatological disorders, and further development of such compounds may be of interest.

Full text of DOI:10.1248/cpb.58.752

752
Note
Chem. Pharm. Bull. 58(5) 752—754 (2010)
Vol. 58, No. 5
Rational Design and Synthesis of 4-O-Substituted
Phenylmethylenethiosemicarbazones as Novel Tyrosinase Inhibitors
Wei YI, Rihui CAO, Zhiyong CHEN, Liang YU, Huan WEN, Qin YAN, Lin MA, and Huacan SONG*
School of Chemistry and Chemical Engineering, Sun Yat-sen University; 135 Xin Gang West Road, Guangzhou 510275,
China. Received January 14, 2010; accepted February 11, 2010; published online February 22, 2010
In continuing our program aimed to search for tyrosinase inhibitors, a series of novel 4-O-substituted
phenylmethylenethiosemicarbazones were rational designed, synthesized and their inhibitory effects on the
diphenolase activity of mushroom tyrosinase were also evaluated. A fair number of compounds were found to
have significant tyrosinase inhibitiory activity. Particularly, the IC50 values of compounds 3a—g, 3j and 3s were
of the same magnitude as tropolone, one of the best tyrosinase inhibitors known so far. Furthermore, the struc-
ture–activity relationships of these compounds were also investigated. All these data suggested that these mole-
cules might be utilized for the development of new candidate for the treatment of dermatological disorders, and
further development of such compounds may be of interest.
Key words synthesis; tyrosinase inhibitory activity; the structure–activity relationship; 4-O-substituted phenylmethyl-
enethiosemicarbazone
Tyrosinase or polyphenol oxidase (EC 1.14.18.1), a multi- inhibitors, and further study suggested that the electron-do-
functional type-3 copper-containing metalloenzyme, is widely nating groups at C-4 are necessary for the actions.14,15) Re-
distributed in nature.1,2) It has a diverse roles in agriculture, cently, our investigation16—19) demonstrated that thiosemicar-
the cosmetic industry and medicine.3) The enzyme catalyzes bzide derivatives exhibited profound tyrosinase inhibitory ac-
two reactions in the melanin biosynthesis pathway involving tivities because of their strong interaction with the dicopper
molecular oxygen: the hydroxylation of monophenols to o- ions active center of tyrosinase. For example, phenylmethyl-
diphenols (cresolase/monophenolase activity) and the oxida- enethiosemicarbazone (Fig. 1) showed a potent tyrosianse
tion of o-diphenols to o-quinones (catecholase/diphenolase inhibitory activity with the IC50 value of 1.93 mM.19) In addi-
activity). The produced quinones are highly reactive and can tion, it was worth noting that, on the basis of the reported
further oxidized spontaneously to form brown pigments of crystallographic structure of tyrosinase, Khatib et al. designed
high molecular weight, namely melanin, which are responsi- and synthesized a series of 3-(2,4-dihydroxyphenyl)propionic
ble for mammalian skin and hair color.3) However, recently acid esters as tyrosinase inhibitors, and the results showed
studies showed that the abnormal accumulation of pigmenta- that these compounds have significant activities against
tion causes the quality loss of vegetables and fruits and vari- tyrosinase.4) This finding emphasized the possibility that a
ous dermatological disorders, such as hyperpigmentation, hydrophobic moiety may also contribute to the inhibition.
freckles, melasma, ephelide, and senile lentigines.4) Further-
Taking advantage of above information, we speculated that
more, the melanin pigments are also found in the mammalian the introduction of a proper hydrophobic subunit to position-
brain where tyrosinase plays a key role in the synthesis of 4 of phenylmethylenethiosemicarbazone, may interact with
neuromelanin and is linked to Parkinson’s and other neurode- the hydrophobic enzyme pocket and the dicopper ions active
generative diseases.4,5) Therefore, tyrosinase inhibitors center of tyrosinase, leading to increased inhibitor-enzyme
should be clinically useful for the treatment of some derma- binding affinity, and thus to an improved compound activity.
tological disorders associated with melanin hyperpigmenta- Therefore, in the present study, a series of 4-O-substituted
tion and also important in cosmetics and food industry.6—8)
phenylmethylenethiosemicarbazones were designed, synthe-
So far, a large number of potential tyrosinase inhibitors sized and their inhibitory effects on the diphenolase activity
have been described from natural source, such as kojic acid of mushroom tyrosinase were evaluated. Moreover, the struc-
(Fig. 1),9) corticosteroids,10) retinoids,10) arbutin,10) tropolone ture–activity relationships of these compounds were also dis-
(Fig. 1).11,12) Unfortunately, only few of these reported com- cussed. We hope that these findings can lead to the discovery
pounds are used in practice because of their lower activities of food preservatives and therapeutically potent agents against
or serious side effects. Therefore, it is still necessary to search clinically dermatological disorders including hyperpigmenta-
and discover novel tyrosinase inhibitors with higher activity tion as well as skin melanoma and offer the efficient informa-
and lower side effect by laboratory synthesis.13)
tions for further designing the tyrosinase inhibitor.
In previous reports, some functionalized 4-alkoxybenz-
aldehyde derivatives have been presented as potent tyrosinase Results and Discussion
The synthesis of compounds 2a—g had been described
detailedly in our previous report.20) The procedure for the
preparation of compounds 2h—t and 3a—t was outlined in
Chart 1. Briefly, compounds 2h—t were synthesized by the
reacting 4-hydroxybenzaldehyde with 3-(bromomethyl)pyri-
dine in anhydrous N,N-dimethylformamide (DMF)/NaH or
the corresponding alkyl bromide in the presence of anhy-
Fig. 1. Structures of Some Known Tyrosinase Inhibitors
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yjhxhc@mail.sysu.edu.cn.

May 2010
753
Reagents and conditions: (i) 2-chloroethanol, 1,4-dioxane, NaOH, H2O, reflux, 2 h; (ii) 2-butoxyethanol or 2-(2-methoxyethoxy)ethanol or 4-methoxybutan-1-ol or 2-
methoxyethanol, NaOH, TsCl, 0 °C, 5 h; K2CO3, THF, reflux, 16 h; (iii) 2-oxo-1,4-butanediol diacetate ester or 2-[(propionyloxy)methyl-hydrogen]-1,3-propane-diol diac-
etate, toluene, 4-methylbenzenesulfonic acid, 100 °C, 0.5 h, then reflux, 10 h; methanol, sodium methoxide, rt, 1 h. epichlorohydrin, 1,4-dioxane, NaOH, H2O, reflux, 2 h;
(iv) the corresponding alkyl bromide, K2CO3, acetone, reflux, 2—5 h; (v) 3-(bromomethyl)pyridine, NaH, DMF, rt, 2 h; (vi) ethanol, thiosemicarbazide, acetic acid, re-
flux, 5—24 h; (vii) ethanol, semicarbazide, acetic acid, reflux, 6 h.
Chart 1. Synthesis of Compounds 2a—t, 3a—t and 4c
Table 1. Inhibitory Effects on Mushroom Tyrosinase of 4-O-Substituted
Phenylmethylenethiosemicarbazones 3a—t and Analogue 4c, Thiosemicar-
bazide, 4-Hydroxybenzaldehyde and Tropolone
drous acetone/K2CO3. Subsequently, the reaction of obtained
compounds 2a—t with thiosemicarbazide and semicarbazide
to give the target compounds 3a—t and 4c, respectively. The
structures of the synthesized compounds were determined by
different spectroscopic techniques, like 1H-NMR and EI-MS,
and purities were confirmed by elemental analysis.21)
Compound IC50 (mmol/l)a)
Compound
IC50 (mmol/l)a)
3a
3b
3c
3d
3e
3f
3g
3h
3i
0.53ꢁ0.092
0.92ꢁ0.109
0.34ꢁ0.054
0.41ꢁ0.065
0.75ꢁ0.063
0.37ꢁ0.082
0.94ꢁ0.133
3.27ꢁ0.335
1.92ꢁ0.159
0.90ꢁ0.098
3.03ꢁ0.168
1.29ꢁ0.112
3m
3n
3o
3p
3q
3r
3s
3t
2.21ꢁ0.281
1.31ꢁ0.132
1.09ꢁ0.165
ꢂ200
ꢂ200
ꢂ200
0.98ꢁ0.059
1.46ꢁ0.236
ꢂ200
The inhibition of our synthetic 4-O-substituted phenyl-
methylenethiosemicarbazones on the diphenolase activity of
mushroom tyrosinase was investigated using the usual proce-
dure22) and compared with tropolone,23) one of the best
tyrosinase inhibitors known so far. The IC50 values of all
obtained compounds were summarized in Table 1. As pre-
dicted, a fair number of compounds were found to have more
potent tyrosinase inhibitiory activity than phenylmethyl-
enethiosemicarbazone (IC50ꢀ1.93 mM). Interestingly, the IC50
values of compounds 3a—g, 3j and 3s were of the same
magnitude as tropolone (IC50ꢀ0.42 mM). Especially, com-
pound 3c bearing a –(CH2)2O(CH2)2OCH3 group was found
to be the most potent inhibitor with the IC50 value of
0.34 mM. However, changing the number of oxygen atom and
the length of alkyl part decreased the inhibitory activity on
4c
3j
3k
3l
Thiosemicarbazideb)
ꢂ2000
4-Hydroxybenzaldehydec) 1220ꢁ52
Tropoloned)
0.42ꢁ0.051
a) Assay performed using mushroom tyrosinase. Values are means of three different
experiments. IC50ꢀmeanꢁS.E.M., S.E.M.: standard error of mean. b) The concen-
tration of 2.00 (mM) corresponding to inhibition percentage, determined in this work, is
39.9ꢁ2.5%. c) Values in the literature8) is 1.20 mM. d) Values in the literature is
0.40 mM.23)
different degree. This might be due to the introduction of elongation of the alkyl chain retarded the binding of inhibitor
other substitution to impede the interaction between this mol- and the active site of tyrosinase, leading to a decrease of
ecule and the enzyme. The results suggested that tyrosinase tyrosinase inhibitiory activity. Moreover, compound 3j
inhibitory activity associated with the configuration of the (IC50ꢀ0.90 mM) displayed 2.1 times higher tyrosinase in-
substitution at the 4-position of benzene, and in the prensent hibitory activity than compound 3i (IC50ꢀ1.92 mM). Com-
investigation, –(CH2)2O(CH2)2OCH3 group represented the pound 3m (IC50ꢀ2.21 mM) showed more potential tyrosinase
most optimal structure for these compounds to exhibit re- inhibitory activity than compound 3k (IC50ꢀ3.03 mM). The
markable tyrosinase inhibitory effects.
results suggested that the introduction of secondary alkyl
In addition, compounds 3i and 3k exhibited the potent ty- group in the side chain might be beneficial to tyrosinase
rosinase inhibitiory activities with IC50 values of 1.92 and inhibitory activity, which supported the previous studies re-
3.03 mM, respectively. Elongation of alkyl chain as in com- ported by Nihei et al.24) Notably, compound 3l (IC50ꢀ1.29
pounds 3n (IC50ꢀ1.31 mM) and 3o (IC50ꢀ1.09 mM) produced mM) bearing the –CH2CHꢀCH2 group in the side chain ex-
slight increase of tyrosinase inhibition, while further elonga- hibited more potential tyrosinase inhibitory activity than 3m,
tion of alkyl chain to give compounds 3p, 3q and 3r, which indicating that the introduction of the –CH2CHꢀCH2 group
showed no tyrosinase inhibitory effect at the concentration of was preferable to –CH(CH3)2 group in inhibition of tyrosi-
200 mM. These results implied that the length of alkyl chain nase. The reason might be that the –CH2CHꢀCH2 moiety
contained in the chain attached on position-4 of phenylmeth- formed the certain interaction with the hydrophobic protein
ylenethiosemicarbazone might be played a very vital role in pocket surrounding the binuclear copper active site.
determining their inhibitory activities, and the excessive
To further expand the structure–activity relationships, com-

754
Vol. 58, No. 5
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pounds 3s and 3t bearing a benzoxyl group were examined
for the tyrosinase inhibitory activity. The results demonstrated
that compounds 3s and 3t also exhibited significant inhibitory
activities with are IC50 values of 0.98 and 1.46 mM, respec-
tively, which are more potent active than 2-(phenylmethylene)-
thiosemicarbazone (IC50ꢀ1.93 mM). However, introducing a
pyridine methyleneoxyl group to position-4 of 2-(phenyl-
methylene)-thiosemicarbazone as in compound 3h (IC50ꢀ
3.27 mM) led to a loss of tyrosinase inhibitory activity. It was
suggested that the nature of aromatic ring might influence the
tyrosinase inhibitory potency, and in the present investiga-
tion, benzoxyl group was much preferable to pyridine meth-
yleneoxyl group in inhibition of tyrosinase. Moreover, when
the thiosemicarbazide moiety of the most active compound
3c was replaced by the semicarbazide group, the obtain com-
pound 4c was inactive at the concentration of 200 mM. The
results showed that the sulfur atom of thiosemicarbazide
moiety was absolutely necessarily for determining the tyrosi-
nase inhibitory activity of these compounds, which futher
supported our previous report.18)
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Conclusion
In this study, a series of 4-O-substituted phenylmethyl-
enethiosemicarbazones were designed, synthesized and eval-
uated as mushroom tyrosinase inhibitors. The results dem-
onstrated that most of compounds had potent tyrosinase
inhibitory activities. Interestingly, the IC50 values of com-
pounds 3a—g, 3j and 3s were of the same magnitude as
tropolone. Especially, compound 3c bearing a –(CH2)2O
(CH2)2OCH3 group was found to be the most potent inhibitor
with the IC50 value of 0.34 mM. Structure–activity relation-
ships (SARs) analysis showed that: (1) Tyrosinase inhibitory
activity associated with the configuration of the substitution
at the 4-position of benzene. (2) The length of alkyl chain
contained in the chain attached on position-4 of phenylmeth-
ylenethiosemicarbazone might be played a very vital role in
determing the inhibitory activity. (3) The introduction of sec-
ondary alkyl group in the side chain might be beneficial to
tyrosinase inhibitory activity. (4) The nature of aromatic ring
might influence the tyrosinase inhibitory potency. (5) The
sulfur atom of thiosemicarbazide moiety was absolutely nec-
essary for determining the tyrosinase inhibitory activity. All
these data suggested that these molecules might be utilized
for the development of new candidate for the treatment of
dermatological disorders. Further modification based on the
current obtained SARs and evaluation of these potent com-
pounds using a human melanoma cell line would be contin-
ued in our laboratory, and the research results will be
reported in due course.
20) Yi W., Cao R. H., Peng W. L., Wen H., Yan Q., Zhou B. H., Ma L.,
Song H. C., Eur. J. Med. Chem., 45, 639—646 (2010).
21) General procedures for the synthesis of compounds 3a—t and 4c: The
appropriate compounds 2a—t (1 mmol) were dissolved in anhydrous
ethanol (15 ml), thiosemicarbazide or semicarbazide (1 mmol) was
added to the above solution. The reaction mixture was refluxed for 5—
24 h and cooled to room temperature. The precipitate was filtered,
washed with ethyl ether, and recrystallized from 95% alcohol to give
compounds 3a—t and 4c in 58—95% yields. Compound 3c (1-(1-(4-
(2-(2-methoxyethoxyl)ethoxyl))benzyliene)thiosemicarbazide): light
orange powder, yield 93%, mp 141—142 °C. IR (KBr, cmꢃ1) n: 3394,
3293, 3158, 3040, 2862, 2851, 1604, 1537, 1259, 1174, 1131, 931,
1
828, 790; H-NMR (DMSO-d6, 300 MHz) d: 11.28 (s, 1H, NH), 8.08
(s, 1H, NH), 7.97 (s, 1H, –CHꢀN–), 7.89 (s, 1H, NH), 7.72 (d, Jꢀ
9.0 Hz, 2H, Ar-H), 6.97 (d, Jꢀ9.0 Hz, 2H, ArH), 4.14 (t, Jꢀ5.0 Hz,
2H, –CH2–), 3.75 (t, Jꢀ4.5Hz, 2H, –CH2–), 3.59(t, Jꢀ4.5 Hz, 2H,
–CH2–), 3.43 (t, Jꢀ3.9 Hz, 2H, –CH2–), 3.23 (s, 3H, –OCH3); 13C-
NMR (DMSO-d6, 75 MHz) d: 178.6, 161.4, 144.4, 129.5, 126.2,
115.4, 72.4, 71.2, 70.0, 67.9, 59.5; ESI-MS: m/z 320 (MꢄNa, 100).
Anal. Calcd: C, 52.51; H, 6.44; N, 14.13. Found: C, 52.54; H, 6.67; N,
14.19.
22) Tyrosinase inhibition assays were performed as follows: Briefly, all the
synthesized compounds were screened for the o-diphenolase inhibitory
activity of tyrosinase using L-DOPA as substrate. All the active in-
hibitors from the preliminary screening were subjected to IC50 studies.
All the synthesized compounds were dissolved in dimethyl sulfoxide
(DMSO) to a concentration of 2.0%. Phosphate buffer pH 6.8 was
used to dilute the DMSO stock solution of test compound. Thirty units
of mushroom tyrosinase (0.2 mg/ml) was first pre-incubated with the
compounds, in 50 mM phosphate buffer (pH 6.8), for 10 min at 25 °C.
Then the L-DOPA (0.5 mM) was added to the reaction mixture and the
enzyme reaction was monitored by measuring the change in absorbance
at 475 nm of the DOPAchrome for 1 min. IC50 value, a concentration
giving 50% inhibition of tyrosinase activity, was determined by inter-
polation of the dose–response curves. Here, 4-hydroxybenzaldehyde
and tropolone were used as the reference inhibitors.
Acknowledgment This work was supported by the Natural Science
Foundation of Guangdong Province, China (2004B30101007).
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