A class of potent tyrosinase inhibitors: Alkylidenethiosemicarbazide compounds

Article abstract of DOI:10.1016/j.ejmech.2008.04.002

A series of alkylidenethiosemicarbazide compounds were synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that most of the synthesized compounds exhibited significant inhibitory activities. Especially, compound 1f was found to be the most potent inhibitor with IC₅₀ value of 0.086 μM, suggesting that further development of such compounds may be of interest.

Full text of DOI:10.1016/j.ejmech.2008.04.002

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1773e1778
http://www.elsevier.com/locate/ejmech
Short communication
A class of potent tyrosinase inhibitors:
Alkylidenethiosemicarbazide compounds
*
Jinbing Liu, Rihui Cao , Wei Yi, Chunming Ma, Yiqian Wan,
*
Binhua Zhou, Lin Ma, Huacan Song
School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
Received 15 October 2007; received in revised form 28 March 2008; accepted 8 April 2008
Available online 27 April 2008
Abstract
A series of alkylidenethiosemicarbazide compounds were synthesized and their inhibitory effects on the diphenolase activity of mushroom
tyrosinase were evaluated. The results showed that most of the synthesized compounds exhibited significant inhibitory activities. Especially,
compound 1f was found to be the most potent inhibitor with IC50 value of 0.086 mM, suggesting that further development of such compounds
may be of interest.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Synthesis; Alkylidenethiosemicarbazide compounds; Tyrosinase inhibitor
1. Introduction
from natural, synthetic and even from semi-synthetic sources,
such as hydroquinone [9], ascorbic acid derivates [10], kojic
acid (Fig. 1) [11], azelaic acid [12], corticosteroids [12], reti-
noids [12], arbutin (Fig. 1) [12] and tropolone (Fig. 1) [13,14].
Among all the known tyrosinase inhibitors, tropolone
(IC50 ¼ 0.4 mM) was found to be one of the most potent tyros-
inase inhibitors [14]. Unfortunately, only few of the reported
compounds are used in medicinal and cosmetic products be-
cause of their lower activities or serious side effects. There-
fore, it is still necessary to search and discover novel
tyrosinase inhibitors with higher activity and lower side effect.
Previous literatures described that thiourea derivatives, such
as phenylthioureas [15,16], alkylthioureas [17] and 1,3-bis-(5-
methanesulfonylbutyl)thiourea, displayed weak or moderate
tyrosinase inhibitory activity. More recently, our investigation
[18] also demonstrated that 1-(1-arylethylidene)thiosemicarb-
zide derivatives exhibited potent inhibitory activities against
mushroom tyrosinase. Stimulated by these results, in the present
investigation, we designed and synthesized a series of novel
alkylidenethiosemicarbazide compounds bearing various alkyl
substituents. We report here the preparation of alkylidenethiose-
micarbazide compounds and their inhibitory effects on the
diphenolase activity of mushroom tyrosinase.
Tyrosinase (EC 1.14.18.1) is a multifunctional copper-
containing enzyme which is widely distributed in plants
and animals [1]. It is well known that tyrosinase can cata-
lyze the hydroxylation of monophenols to o-diphenols
(monophenolase activity) and the oxidation of o-diphenols
to o-quinone (diphenolase activity). And the enzymatic
oxidation of L-tyrosine into melanin is of considerable
importance in coloring of skin, hair and eyes, and in
food browning [2,3]. In addition, tyrosinase is also related
to the molting process of insects [4] and adhesion of
marine organisms [3,5].
Nowadays, tyrosinase inhibitors are thought to be clinically
useful for the treatment of some dermatological disorders
associated with melanin hyperpigmentation [6,7], and useful
in cosmetic products and food industry [8]. So far, a large
number of potential tyrosinase inhibitors have been discovered
* Corresponding authors. Tel.: þ86 20 84110918; fax: þ86 20 84112245.
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), yjhxhc@mail.sysu.
edu.cn (H. Song).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.002

1774
J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1773e1778
O
O
OH
OH
HO
OH
O
OH
O
H3CO
CH=CHCO2H
HO
HO
O
OH
arbutin
4-methoxycinnamic acid
tropolone
kojic acid
Fig. 1. Chemical structure of known tyrosinase inhibitors.
2. Chemistry
suggested that the double bonds might be detrimental to
their inhibitory activities.
The alkylidenethiosemicarbazides were prepared by the
condensation reaction of alkyl ketones or aldehydes with
thiosemicarbazide in the presence of acetic acid in ethanol.
To investigate the effects of thiosemicarbazido group on tyros-
inase inhibitory activity, pentane-2,4-dione, hexane-2,5-dione,
2-acetylcyclohexanone and cyclohexane-1,3-dione were
selected to prepare dithiosemicarbazide compounds. Unfortu-
nately, only mono-thiosemicarbazido group products were ob-
tained (Scheme 1).
(5) Compound 3 bearing a carbonyl group was also less active
than the corresponding thiosemicarbazides without a car-
bonyl group. These results indicated that electron-
withdrawing effects of the carbonyl group might play a vi-
tal role in determining their inhibitory activities.
4. Conclusions
The present investigation reported that, for the first time,
alkylidenethiosemicarbazide compounds had potent inhibitory
effects on the diphenolase activity of mushroom tyrosinase.
Interestingly, compound 1f was found to be the most potent in-
hibitor with IC50 value of 0.086 mM. Preliminary structuree
activity relationships’ (SARs) analysis indicated that (1) the
increase of the length of alkyl chain might cause stereo-
hindrance for the inhibitors approaching the active site of
the enzyme resulting in the decrease of inhibitory activities,
and the shorter alkyl chain was more favorable; (2) the double
bonds might be detrimental to their activities; and (3) the
electron-withdrawing substituents might play a vital role in
determining their inhibitory activities. These results suggested
that further development of such compounds may be of
interest.
3. Biology
Taking all the newly synthesized compounds as the effec-
tors, we investigated their inhibitory effects on the diphenolase
activity of mushroom tyrosinase. The IC50 value of these com-
pounds are summarized in Table 1.
From the data shown in Table 1, the following conclusions
were drawn.
(1) All the compounds exhibited potent inhibitory effects on
the diphenolase activity of mushroom tyrosinase. Particu-
larly,
compounds
1a
(IC50 ¼ 0.23 mM),
1b
(IC50 ¼ 0.20 mM), 1f (IC50 ¼ 0.086 mM) and 1g
(IC50 ¼ 0.28 mM) demonstrated more potent inhibitory
activities than reference standard inhibitors arbutin (30%
inhibition at 10.4 mM) and 3-(4-methoxyphenyl)acrylic
acid (IC50 ¼ 0.41 mM), and the most representative inhib-
itor, tropolone (IC50 ¼ 0.4 mM), described by the literature
[13,14].
5. Experimental protocols
5.1. Reagents and general procedures
(2) With the increase of the length of chain R (for 1-alkylide-
nethiosemicarbazides 1aee and 1-(1-alkylethylidene)thio-
semicarbazides 1fek), the inhibitory activities decreased
gradually. Interestingly, compound 1f bearing a methyl
group was found to be the most potent inhibitor with an
IC50 value of 0.086 mM. These results suggested that the
increase of the length of alkyl chain might cause stereo-
hindrance for the inhibitors approaching the active site
of the enzyme.
(3) Compound 2a was 5.5-fold more active than compound
2b, which indicated that the smaller size of the ring in
volume would be beneficial to the molecules to approach
the active centre of the enzyme. These results further
confirmed that the shorter chain R was more favorable
for the inhibitory activities of obtained compounds.
(4) The synthesized compounds with a saturated R substitu-
ents displayed stronger inhibitory activities than those
compounds with an unsaturated R substituents, which
Melting points (m.p.) were determined with WRS-1B melt-
ing point apparatus and the thermometer was uncorrected.
NMR spectra were recorded on Mercury-Plus 300 spectrome-
ters at 25 ꢀC in CDCl3 or DMSO-d6. All chemical shifts (d)
are quoted in parts per million downfield from TMS and
coupling constants (J ) are given in hertz. LCeMS spectra
were recorded using the LCMS-2010A. All reactions were
monitored by TLC (Merck Kieselgel 60 F254) and the spots
were visualized under UV light. Elemental analyses were per-
formed on a Vario EL instrument and were within ꢁ0.4% of
the theoretical values. Infrared (IR) spectra were recorded on
VECTOR 22 spectrometer. The appropriate aldehyde, ketone,
thiosemicarbazide, and 4-methoxycinnamic acid were pur-
chased from Darui Chemical Co. (ShangHai, China), arbutin
was obtained from Brillian Biochemical Co. The other com-
mercially available reagents and solvents were used without
further purification. Mushroom tyrosinase (specific activity

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1773e1778
1775
S
S
NNHCNH2
NH2NHCNH2
R
H
R
O
H
Methanol, reflux
1a-1e
1a, R = CH3; 1b, R = C2H5; 1c, R = Iso-C3H7; 1d, R = CH3CH=CH; 1e, R = PhCH=CH
S
S
NNHCNH2
NH2NHCNH2
R
R
O
H3C
H3C
Methanol, reflux
1f-1j
1f, R = CH3; 1g, R = C2H5; 1h, R = CH=C(CH3)2; 1i, R = 4-CH3OC6H4CH2;
1j, R2 = 4-HOC6H4CH2CH2
S
S
(
)n
(
)n
NH2NHCNH2
O
NNHCNH2
Methanol, reflux
2a - 2b
2a. n=1; 2b. n=2
S
S
NH2NHCNH2
Methanol, reflux
O
NNHCNH2
O
O
3
S
O
NH2NHCNH2
Methanol, reflux
CH3
CH3
H3C
N
H3C
NHCNH2
O
S
4
Scheme 1. Synthesis of alkylidenethiosemicarbazide compounds.
of the enzyme is 5360 U/mg) and L-DOPA (L-3,4-dihydroxy-
phenylalanine) were purchased from Sigma Chemical Co.
5.2.1. 1-Ethylidenethiosemicarbazide (1a)
M.p. 135e136 ꢀC. Rf ¼ 0.58 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
d 11.01 (1H, br s, NH), 7.90 (1H, br s, NH2), 7.43 (1H, br s,
NH2), 7.37 (H, q, J ¼ 5.4 Hz, CH), 1.84 (3H, d, J ¼ 5.4 Hz,
CH3). 13C NMR (75 MHz, DMSO-d6): d 177.9, 144.5, 19.0.
IR (KBr): 3379, 3261, 3189, 3024, 1591, 1535 cmꢂ1. MS
(ESI): m/z (100%) ¼ 118 (M þ 1). Anal. Calcd for C3H7N3S
(117.17): C, 30.75; H, 6.02; N, 35.86. Found: C, 30.84; H,
5.99; N, 35.98.
5.2. General procedures for the synthesis of
alkylidenethiosemicarbazide compounds
The appropriate aldehyde or ketone (10 mmol) was dis-
solved in anhydrous ethanol (10 mL), and thiosemicarbazide
(10 mmol) and acetic acid (0.5 mL) were added into the solu-
tion. After being refluxed for 24 h, the reaction mixture was
cooled to room temperature and then the precipitate appeared.
The corresponding pure compounds were obtained by filtra-
tion. For some special cases, the target compounds could be
purified by recrystallization from ethanol.
5.2.2. 1-Propylidenethiosemicarbazide (1b)
M.p. 150e152 ꢀC. Rf ¼ 0.62 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):

1776
J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1773e1778
Table 1
Tyrosinase inhibitory activities of alkylidenethiosemicarbazide compounds
d, J ¼ 9.0 Hz, CH), 7.58 (1H, br s, NH2), 7.55 (2H, d,
J ¼ 7.2 Hz, phH), 7.32e7.37 (3H, m, phH), 7.03 (1H, d,
J ¼ 16.2 Hz, CH), 6.89 (1H, t, J ¼ 9.3 Hz, CH). 13C NMR
(75 MHz, CDCl3): d 178.3, 145.4, 139.5, 136.5, 129.8,
129.5, 127.6, 125.7. IR (KBr): 3398, 3279, 3152, 3028,
1601, 757 cmꢂ1. MS (ESI): m/z (100%) ¼ 206 (M þ 1).
Compounds
Yield (%)
IC50 (mM) (mean ꢁ SEMa)
1a
1b
89.2
83.6
85.1
81.5
76.2
91.5
88.3
53.6
68.1
56.8
78.3
74.6
51.0
46.8
0.23 ꢁ 0.015
0.20 ꢁ 0.018
1c
1d
0.62 ꢁ 0.05
1.00 ꢁ 0.026
1e
2.70 ꢁ 0.058
5.2.6. 1-(Propan-2-ylidene)thiosemicarbazide (1f)
1f
1g
0.086 ꢁ 0.009
0.28 ꢁ 0.023
M.p. 180e181 ꢀC. Rf ¼ 0.55 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
1h
1i
11.50 ꢁ 0.13
0.42 ꢁ 0.031
d 9.86 (1H, br s, NH), 7.95 (1H, br s, NH2), 7.48 (1H, br s,
NH2), 1.91 (3H, s, CH3), 1.89 (3H, s, CH3). 13C NMR
(75 MHz, DMSO-d6): d 178.9, 152.3, 25.9, 18.4. IR (KBr):
3378, 3234, 3152, 2997, 1596, 1512 cmꢂ1. MS (ESI): m/z
(100%) ¼ 132 (M þ 1). Anal. Calcd for C4H9N3S (131.20):
C, 36.62; H, 6.91; N, 32.03. Found: C, 36.59; H, 6.84; N, 32.25.
1j
2a
0.54 ꢁ 0.012
0.17 ꢁ 0.011
2b
3
0.95 ꢁ 0.032
15.10 ꢁ 0.030
0.85 ꢁ 0.064
4
4-Methoxycinnamic acidb
0.41 (mM) ꢁ 0.005
10.40 (mM) ꢁ 0.089
Arbutinc
a
SEM: standard error of the mean.
IC50 values in the literature is 0.34e0.43 mM [20,21].
The concentration of 10.4 mM corresponding to inhibition percentage, de-
5.2.7. 1-(Butan-2-ylidene)thiosemicarbazide (1g)
b
c
M.p. 98e100 ꢀC. Rf ¼ 0.65 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
termined in this work, is 30%. The reported IC50 value of arbutin was more
than 30 mM [22].
d 8.58 (1H, br s, NH), 7.26 (1H, br s, NH2), 6.45 (1H, br s,
NH2), 2.31e2.33 (2H, m, CH2), 1.90 (3H, s, CH3), 1.10
(3H, t, J ¼ 7.6 Hz, CH3). 13C NMR (75 MHz, DMSO-d6):
d 179.1, 155.9, 32.3, 17.1, 11.4. IR (KBr): 3381, 3236,
3150, 3019, 2971, 1578 cmꢂ1. MS (ESI): m/z (100%) ¼ 146
(M þ 1). Anal. Calcd for C5H11N3S (145.23): C, 41.35; H,
7.63; N, 28.93. Found: C, 41.17; H, 7.55; N, 28.71.
d 10.99 (1H, br s, NH), 7.89 (1H, br s, NH2), 7.39 (1H, br s,
NH2), 7.38 (1H, t, J ¼ 3.8 Hz, CH), 2.17e2.20 (2H, m,
CH2), 0.97 (3H, t, J ¼ 4.8 Hz, CH3). 13C NMR (75 MHz,
DMSO-d6): d 178.1, 149.0, 26.0, 11.3. IR (KBr): 3385,
3273, 3189, 3036, 1586, 1541 cmꢂ1
. MS (ESI): m/z
(100%) ¼ 132 (M þ 1). Anal. Calcd for C4H9N3S (131.20):
C, 36.62; H, 6.91; N, 32.03. Found: C, 36.84; H, 6.97; N,
32.22.
5.2.8. 1-(4-Methylpent-3-en-2-ylidene)
thiosemicarbazide (1h)
M.p. 119e120 ꢀC. Rf ¼ 0.70 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
5.2.3. 1-(3-Methylbutylidene)thiosemicarbazide (1c)
d 9.76 (1H, br s, NH), 8.09 (1H, br s, NH2), 7.55 (1H, br s,
NH2), 4.74 (1H, s, CH), 1.94 (3H, s, CH3), 0.99 (6H, s,
2CH3). 13C NMR (75 MHz, DMSO-d6): d 179.1, 153.5,
152.9, 136.7, 25.9, 20.1. IR (KBr): 3385, 3263, 3158, 3025,
2961, 1385 cmꢂ1. MS (ESI): m/z (100%) ¼ 172 (M þ 1).
Rf ¼ 0.73 (ethyl acetate/petroleum ether (bp 60e
1
90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, CDCl3): d 9.86 (1H,
br s, NH), 7.28 (1H, br s, NH2), 7.08 (1H, br s, NH2), 6.48
(1H, t, J ¼ 6.3 Hz, CH), 2.10 (2H, t, J ¼ 6.0 Hz, CH2),
1.88e1.91 (1H, m, CH), 0.97 (6H, d, J ¼ 6.3 Hz, 2CH3). 13C
NMR (75 MHz, CDCl3): d 177.6, 148.8, 41.4, 26.9, 22.8. IR
(KBr): 3425, 3282, 3144, 2951, 1590, 1537, 1383 cmꢂ1. MS
(ESI): m/z (100%) ¼ 160 (M þ 1).
5.2.9. 1-(1-(4-Methoxyphenyl)propan-2-ylidene)-
thiosemicarbazide (1i)
M.p. 122e123 ꢀC. Rf ¼ 0.68 (ethyl acetate/petroleum ether
(bp 60e90 ꢀC) ¼ 2:1, v/v). 1H NMR (300 MHz, CDCl3):
d 8.50 (1H, br s, NH), 7.25 (1H, br s, NH2), 7.09 (2H, d,
J ¼ 8.7 Hz, phH), 6.86 (2H, d, J ¼ 8.7 Hz, phH), 6.34 (1H,
br s, NH2), 3.80 (3H, s, CH3), 3.50 (2H, s, CH2), 1.81 (3H,
s, CH3). 13C NMR (75 MHz, CDCl3): d 180.2, 157.6, 155.7,
131.2, 130.5, 114.5, 55.7, 38.6, 14.5. IR (KBr): 3405, 3210,
3137, 3031, 2833, 1587, 1514, 859 cmꢂ1. MS (ESI): m/z
(100%) ¼ 238 (M þ 1). Anal. Calcd for C11H15N3OS
(237.32): C, 55.67; H, 6.37; N, 17.71. Found: C, 55.61; H,
6.32; N, 17.76.
5.2.4. 1-(But-2-enylidene)thiosemicarbazide (1d)
M.p. 142e143 ꢀC. Rf ¼ 0.73 (ethyl acetate/petroleum ether
(bp 60e90 ꢀC) ¼ 2:1, v/v). 1H NMR (300 MHz, CDCl3):
d 9.70 (1H, br s, NH), 7.53 (1H, d, J ¼ 7.8 Hz, CH), 7.06
(1H, br s, NH2), 6.37 (1H, br s, NH2), 6.13e6.18 (2H, m,
2CH), 1.91 (3H, d, J ¼ 4.5 Hz, CH3). 13C NMR (75 MHz,
CDCl3): d 178.2, 145.6, 138.9, 129.1, 19.3. IR (KBr): 3384,
3275, 3163, 3021, 2961, 1585, 1527 cmꢂ1. MS (ESI): m/z
(100%) ¼ 144 (M þ 1). Anal. Calcd for C5H9N3S (143.21):
C, 41.93; H, 6.33; N, 29.34. Found: C, 41.84; H, 6.18; N, 29.68.
5.2.10. 1-(4-(4-Hydroxyphenyl)butan-2-ylidene)-
thiosemicarbazide (1j)
5.2.5. 1-(3-Phenylallylidene)thiosemicarbazide (1e)
M.p. 134e136 ꢀC. Rf ¼ 0.75 (ethyl acetate/petroleum ether
(bp 60e90 ꢀC) ¼ 2:1, v/v). 1H NMR (300 MHz, CDCl3):
d 11.36 (1H, br s, NH), 8.15 (1H, br s, NH2), 7.87 (1H,
M.p. 154e155 ꢀC. Rf ¼ 0.69 (ethyl acetate/petroleum ether (bp
1
60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6): d 8.49
(1H, br s, NH), 7.12 (1H, br s, NH2), 7.02 (2H, d, J ¼ 6.3 Hz,

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1773e1778
1777
phH), 6.77 (2H, d, J ¼ 6.3 Hz, phH), 6.22 (1H, br s, NH2), 2.80
6. Tyrosinase assay
(2H, t, J ¼ 7.2 Hz, CH2), 2.57 (2H, t, J ¼ 7.2 Hz, CH2), 1.89
(3H, s, CH3). 13C NMR (75 MHz, DMSO-d6): d 179.1,
159.3, 148.9, 129.1, 128.8, 115.7, 32.5, 29.3, 19.8. IR (KBr):
3609, 3366, 3265, 3187, 1610, 1513, 826 cmꢂ1. MS (ESI):
m/z (100%) ¼ 236 (M ꢂ 1). Anal. Calcd for C11H15N3OS
(237.32): C, 55.67; H, 6.37; N, 17.71. Found: C, 55.73; H,
6.35; N, 17.79.
Tyrosinase inhibition assays were performed according to
the method developed by Hearing [19] with slight modifica-
tion. Briefly, all the synthesized compounds were screened
for the o-diphenolase inhibitory activity of tyrosinase using
L-DOPA as substrate. All the active inhibitors from the prelim-
inary screening were subjected to IC50 studies. All the synthe-
sized compounds were dissolved in 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 mush-
room tyrosinase (0.2 mg/mL) were first pre-incubated with the
compounds, in 50 mM phosphate buffer (pH 6.8), for 10 min
at 25 ꢀC. Then 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 for formation of the
DOPA chrome for 1 min. Doseeresponse curves were ob-
tained by performing assays in the presence of increasing con-
centrations of inhibitors (at least eight concentrations). IC50
value, a concentration giving 50% inhibition of tyrosinase ac-
tivity, was determined by interpolation of the doseeresponse
curves. The percent of inhibition of tyrosinase reaction was
calculated as following:
5.2.11. 1-Cyclopentylidenethiosemicarbazide (2a)
M.p. 152e154 ꢀC. Rf ¼ 0.65 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
d 8.45 (1H, br s, NH), 7.16 (1H, br s, NH2), 6.44 (1H, br s,
NH2), 2.39 (2H, t, J ¼ 6.6 Hz, CH2), 2.31 (2H, t, J ¼ 6.6 Hz,
CH2), 1.89e1.91 (2H, m, CH2), 1.78e1.80 (2H, m, CH2).
13C NMR (75 MHz, DMSO-d6): d 178.5, 164.0, 33.7, 28.5,
25.3. IR (KBr): 3383, 3261, 3138, 2957, 1661 cmꢂ1. MS
(ESI): m/z (100%) ¼ 158 (M þ 1). Anal. Calcd for
C6H11N3S (157.24): C, 45.83; H, 7.05; N, 26.72. Found: C,
45.95; H, 7.09; N, 26.85.
Inhibition rateð%Þ ¼ ½ðB ꢂ SÞ=Bꢃ ꢄ 100
5.2.12. 1-Cyclohexylidenethiosemicarbazide (2b)
M.p. 154e155 ꢀC. Rf ¼ 0.65 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
Here, the B and S are the absorbances for the blank and sam-
ples, respectively. 4-Methoxycinnamic acid and arbutin were
used as reference standard inhibitors for comparison. All the
studies were carried out at least in triplicate and the results
here represent mean ꢁ SEM (standard error of the mean).
d 8.82 (1H, br s, NH), 7.26 (1H, br s, NH2), 6.46 (1H, br s,
NH2), 2.25e2.33 (4H, m, 2CH2), 1.63e1.71 (6H, m, 3CH2).
13C NMR (75 MHz, DMSO-d6): d 178.8, 157.5, 27.4, 26.3,
25.8. IR (KBr): 3379, 3217, 3144, 2939, 2858, 1585 cmꢂ1
.
MS (ESI): m/z (100%) ¼ 172 (M þ 1). Anal. Calcd for
C7H13N3S (171.26): C, 49.09; H, 7.65; N, 24.54. Found: C,
49.06; H, 7.63; N, 24.75.
Acknowledgment
This work was supported by the Natural Science Founda-
tion of Guangdong Province, China (2004B30101007).
5.2.13. 1-(3-Oxocyclohexylidene)thiosemicarbazide (3)
M.p. 175e176 ꢀC. Rf ¼ 0.66 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
d 11.85 (1H, br s, NH), 9.20 (1H, br s, NH2), 7.38 (1H, br s,
NH2), 3.42 (2H, s, CH2), 2.21e2.41 (4H, m, 2CH2), 1.71e
1.72 (2H, m, CH2). 13C NMR (75 MHz, DMSO-d6):
d 208.7, 178.6, 159.2, 46.8, 44.7, 22.5, 19.2. IR (KBr):
References
[1] K.K. Song, H. Huang, P. Han, C.L. Zhang, Y. Shi, Q.X. Chen, Biochem.
Biophys. Res. Commun. 342 (2006) 1147e1151.
3416, 3275, 3161, 1716, 1603 cmꢂ1
.
MS (ESI): m/z
[2] M. Perez-Gilabert, F. Garcıa-Carmona, Biochem. Biophys. Res. Com-
´
mun. 285 (2001) 257e261.
´
(100%) ¼ 186 (M þ 1).
[3] S. Okombi, D. Rival, S. Bonnet, A.M. Mariotte, E. Perrier,
A. Boumendjel, Bioorg. Med. Chem. Lett. 16 (2006) 2252e2255.
[4] M. Sugumaran, FEBS Lett. 293 (1991) 4e10.
[5] K. Marumo, J.H. Waite, Biochim. Biophys. Acta 872 (1986) 98e103.
[6] M. Shiino, Y. Watanabe, K. Umezawa, Bioorg. Med. Chem. 9 (2001)
1233e1240.
5.2.14. 1-(2,5-Dimethyl-1H-pyrrol-1-yl)thiourea (4)
M.p. 217e218 ꢀC. Rf ¼ 0.75 (ethyl acetate/petroleum ether
1
(bp 60e90 ꢀC) ¼ 2:1, v/v). H NMR (300 MHz, DMSO-d6):
[7] A. Palumbo, G. Misuraco, G. Prota, Biochim. Biophys. Acta 1073 (1991)
85e90.
d 10.19 (1H, br s, NH), 8.18 (1H, br s, NH2), 6.54 (1H, br s,
NH2), 5.68 (2H, s, 2CH), 1.99 (6H, s, 2CH3). 13C NMR
(75 MHz, DMSO-d6): d 183.15, 127.64, 104.71, 11.73. IR
(KBr): 3385, 3231, 3146, 1956, 1616 cmꢂ1. MS (ESI): m/z
(100%) ¼ 170 (M þ 1). Anal. Calcd for C7H11N3S (169.25):
C, 49.68; H, 6.55; N, 24.83. Found: C, 49.59; H, 6.51; N, 25.21.
[8] K.M. Khan, G.M. Maharvi, M.T.H. Khan, A.J. Shaikh,
S. Perveen, S. Begum, M.I. Choudhary, Bioorg. Med. Chem. 14
(2006) 344e351.
[9] A. Garcia, J.E. Fulrton, Dermatol. Surg. 22 (1996) 443e447.
[10] S. Kojima, H. Yamaguch, K. Morita, Y. Ueno, R. Paolo, Biol. Pharm.
Bull. 18 (1995) 1076e1080.

1778
J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1773e1778
´
[11] J. Cabanes, S. Chazarra, F. Garcıa-Carmona, J. Pharm. Pharmacol. 46
(1994) 982e985.
[16] M. Criton. FR Patent 2880022, June 26, 2006.
[17] J. Daniel. U.S. Patent 2006135618, June 22, 2006.
[18] J. Liu, W. Yi, Y. Wan, L. Ma, H. Song, Bioorg. Med. Chem. 16 (2008)
1096e1102.
ˇ
[12] G.M. Casanola-Martın, M.T.H. Khan, Y. Marrero-Ponce, A. Ather,
´
M.N. Sultankhodzhaev, F. Torrens, Bioorg. Med. Chem. Lett. 16
(2006) 324e330.
[13] S.M. Son, K.D. Moon, C.Y. Lee, J. Agric. Food Chem. 48 (2000) 2071e
2074.
[19] V.J. Hearing, Methods in Enzymology, vol. 142, Academic Press, New
York, 1987, p 154.
[20] Y. Shi, Q.X. Chen, Q. Wang, K.K. Song, L. Qiu, Food Chem. 92 (2005)
707e712.
[21] H.S. Lee, J. Agric. Food Chem. 50 (2002) 1400e1403.
[22] S. Kazuhisa, N. Koji, N. Takahisa, K. Taro, S. Kenji, J. Biosci. Bioeng.
99 (2005) 272e276.
[14] K. Iida, K. Hase, K. Shimomura, S. Sudo, S. Kadota, Planta Med. 61
(1995) 425e428.
[15] T. Klabunde, C. Eicken, J.C. Sacchettini, Nat. Struct. Biol. 5 (1998)
1084e1090.