Analogues of N-hydroxy-N′-phenylthiourea and N-hydroxy-N′-phenylurea as inhibitors of tyrosinase and melanin formation

Article abstract of DOI:10.1016/j.bmcl.2008.04.079

A series of N-hydroxy-N′-phenylthiourea and N-hydroxy-N′-phenylurea analogues were prepared and evaluated as inhibitors of tyrosinase and melanin formation. The most active analogue 1 inhibited mushroom tyrosinase with an IC₅₀ of around 0.29 μM

Full text of DOI:10.1016/j.bmcl.2008.04.079

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3607–3610
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Analogues of N-hydroxy-N0-phenylthiourea and N-hydroxy-
N0-phenylurea as inhibitors of tyrosinase and melanin formation
*
Marc Criton, Véronique Le Mellay-Hamon
Mayoly Spindler S.A. Laboratory, Preclinical R&D, 6, avenue de l’Europe—BP51, 78401 Chatou cedex, France
a r t i c l e i n f o
a b s t r a c t
A series of N-hydroxy-N0-phenylthiourea and N-hydroxy-N0-phenylurea analogues were prepared and
evaluated as inhibitors of tyrosinase and melanin formation. The most active analogue 1 inhibited mush-
room tyrosinase with an IC50 of around 0.29 lM and also retained a substantial potency in cell culture by
reducing pigment synthesis by 78%. Therefore, compound 1 could be considered as a promising candidate
for preclinical drug development for skin hyperpigmentation application.
Article history:
Received 7 April 2008
revised 29 April 2008
Accepted 29 April 2008
Available online 4 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Melanin
Tyrosinase inhibitors
Phenylthiourea
N-Hydroxy-N0-phenylthiourea and
N-hydroxy-N0-phenylurea derivatives
Mammals skin pigmentation results from the production of
melanin by melanocytes and its accumulation in the epidermis.
Melanin synthesis or melanogenesis is a complex pathway involv-
ing enzymatic and chemical reactions, which are restricted to mel-
anosomes, melanocyte-specific organelles containing all
components required to synthesise pigment. Among enzymes in-
volved in melanin biosynthesis tyrosinase, a copper-containing,
membrane-bound glycoprotein is the most critical and rate-limit-
ing enzyme that catalyzes the first two steps in the biosynthetic
N-Phenylthiourea (PTU, Fig. 1) was shown to inhibit catechol oxi-
dase enzyme that belongs with tyrosinase to the type-3 copper
proteins group. The sulfur atom of the PTU binds to both copper
ions in the active site of catechol oxidase and blocks enzyme activ-
ity.14,15 Besides, our interest for chelators agents led us to study the
hydroxamic acid group. Hydroxamate molecules, one of the major
classes of naturally occurring metal complexing agents, have been
thoroughly studied as ligands for different metal ions as Fe(III),
Zn(II), and Cu(II).16,17 The chelation involves the oxygen belonging
to the carbonyl moiety and the NHOH groups. Numerous papers
pathway: hydroxylation of tyrosine to
-DOPA) and oxidation of -DOPA to dopaquinone.
Increased production and accumulation of melanin characterize
L-dihydroxyphenylalanine
(L
L
a large number of dermatological disorders, which include ac-
quired hyperpigmentation, such as melasma, freckles, postinflam-
matory melanoderma, and solar lentigo.1,2 Many tyrosinase
inhibitors find application in cosmetics and pharmaceutical prod-
ucts as a way of preventing overproduction of melanin in epider-
mis. Hydroquinone is one of the most potent whitening agents
first discovered,3,4 but since its introduction some adverse effects
have been recognized. In recent years various tyrosinase inhibitors
have been reported such as azelaic acid,5 ascorbic acid derivatives,6
arbutin,7 kojic acid,8 hydroxystilbene compounds like resvera-
trol,9–11 and methyl ester of gentisic acid.12 Most of the tyrosinase
inhibitors are phenol/catechol derivatives, structurally similar to
H
N
H
N
NH2
NH2
S
O
N-phenylthiourea (PTU)
N-phenylurea
H
N
H
N
R
H2N
OH
OH
tyrosine or L
-DOPA, which act as suicide substrates of tyrosinase.13
O
O
N-hydroxyurea
Hydroxamic acid (R = alkyl, Bn)
* Corresponding author. Tel.: +33(0)134805583; fax: +33(0)134805576.
E-mail address: veronique.lemellay-hamon@mayoly-spindler.fr (V. Le Mellay-
Hamon).
Figure 1. Chemical structures of N-phenylurea, N-phenylthiourea, hydroxamic
acid, and hydroxyurea.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.079

3608
M. Criton, V. Le Mellay-Hamon / Bioorg. Med. Chem. Lett. 18 (2008) 3607–3610
showed that N-hydroxyurea (–NHC(O)NHOH) function seems par-
ticularly suited as hydroxamate alternative in metal-chelating abil-
ity since it incorporates the C(O)–NHOH group that is necessary to
establish the same ideal type of metal chelation. N-Hydroxyurea it-
self, used as antineoplastic drug since 1960s,18 is known to form a
complex with Fe(III) and Cu(II) metal ions.19
In the present report, using PTU as starting point, we designed
and synthesized new compounds where the primary amino group
of PTU was replaced by hydroxylamino derivatives and the sulfur
atom was conserved or replaced by an oxygen atom to increase
Cu2+-chelating properties of the resulting compounds, thereby
generating N-hydroxy-N0-phenylthiourea and N-hydroxy-N0-phe-
nylurea derivatives (1–22, Tables 1–3). These compounds were
evaluated on tyrosinase activity in vitro and on melanin produc-
tion by cultured melanocytes.
1, a number of commercially available phenylisocyanate or phenyl-
isothiocyanate were treated with different N-hydroxylamine
derivatives in the presence of dimethylformamide, and triethyl-
amine affording N-hydroxy-N0-phenylthiourea and N-hydroxy-
N0-phenylurea derivatives (1–14; 18–22). Compounds (15–17)
were prepared according to Scheme 2, by treating phenylcarba-
moyl chloride derivatives with N-hydroxylamine derivatives in
the presence of dimethylformamide, dichoromethane and
triethylamine.21,22
Then all these derivatives were evaluated on mushroom tyros-
inase activity and their ability to inhibit melanin formation by B16
melanoma cell line was investigated. Consistent with previous re-
ports,23,24 PTU induced a strong inhibition of the tyrosinase activity
(IC50 = 1.8 lM) in contrast to N-phenylurea which showed no inhi-
bition in our assay (Table 1). Interestingly, when the amino group
and the sulfur moieties of the PTU were replaced by N-hydroxyl-
amine and oxygen, respectively, the resulting compound 1 was
more potent to inhibit tyrosinase activity (IC50 = 0.29 lM) com-
pared to PTU. Besides, tyrosinase inhibition with compound 1
was more potent than that obtained with the reference tyrosinase
inhibitors, kojic acid and hydroquinone, for which the IC50 values
were, respectively, 75 and 37 lM.
Thus compound 1 was used as benchmark compound to synthe-
size a series of derivatives in which different substitutions were
introduced at the N-hydroxyurea moiety (–NH–CO–NHOH) while
keeping the phenyl ring unmodified (Table 1). When the terminal
NHOH group was methylated on the hydroxyl (13, 17) or on the
NH (14, 16) moiety, the tyrosinase activity was completely lost.
Methylation on N0 leads to diminished tyrosinase activity, but
when NHOH motif was conserved (15) the product had a more po-
tent inhibition (IC50 = 16 lM) than kojic acid and hydroquinone,
but lower than compounds 1 and PTU. When the hydroxyl of the
NHOH moiety was silylated with tert-butyldimethylsilyl group
the resulting compound 22 (IC50 = 70 lM) showed an inhibitory
activity comparable to kojic acid. Acetylation of the R0 position
(compound 18) had a low inhibitory activity (IC50 value of
170 lM) compared to 1.
The detailed chemistry of compounds has been previously de-
scribed by our group in a patent.20 Briefly, and according to Scheme
Table 1
Structures (1, 13–18, 22), tyrosinase, and melanin-formation inhibition
Compound
Substituent
R0
Tyrosinase Melanin
inhibition
production
R
R00
IC50 (lM)
% Inhibition
R''
N
R'
N
at 100 lMa
R
O
1
OH
OMe
OH
OH
OH
OMe
OH
OTBDMSi
H
H
Me
H
H
H
H
0.29
>1000
>1000
78 2.12
ni
ni
nd
nd
nd
nd
ni
13
14
15
16
17
18
22
Me 16
Me Me >1000
H
Ac
H
Me >1000
H
H
170
70
N-Phenylthiourea
N-Phenylurea
Kojic acid
Hydroquinone
Arbutin
1.8
>1000
75
37
—
58 0.77
ni
54.3 1.09b
—
With the exception of the compound 22, our results indicate
that an unsubstituted NHOH moiety is important for inhibition of
the tyrosinase activity, suggesting that the chelating ability of
N-hydroxyurea might be important for a potent inhibition of tyros-
43.8 0.15
a
Results are represented as inhibition %, means SE of three independent tests.
Kojic acid was tested at 1 mM; ni, no inhibition; nd, not done.
b
Table 2
Structures (1–12), tyrosinase and melanin-formation inhibition
Compound
Substituent
R4
Tyrosinase inhibition
Melanin production
R0
R2
IC50 (lM)
% Inhibition at 100 lMa
R2
R'
N
H
N
OH
O
R4
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
Me
H
H
OH
H
H
H
OMe
H
H
H
H
H
0.29
41
32
>1000
>1000
6.3
2.6
770
27
4.3
2.7
78 2.12
19 1.31
ni
ni
ni
OMe
OMe
OBu
OBn
NO2
NO2
NHCONHOH
CF3
ni
86 0.44
82 0.23
66 1.54
79.3 0.58
75.5 2.30
ni
10
11
12
H
H
Me
H
H
H
Br
Br
>1000
N-Phenylthiourea, N-phenylurea, kojic acid, hydroquinone, arbutin
See Table 1
a
Results are represented as inhibition %, means SE of three independent tests; ni, no inhibition.

M. Criton, V. Le Mellay-Hamon / Bioorg. Med. Chem. Lett. 18 (2008) 3607–3610
3609
Table 3
Structures (19–21), tyrosinase and melanin-formation inhibition
Compound
Substituent
R4
Tyrosinase inhibition
Melanin production
R
R3
IC50 (lM)
% Inhibition at 100 lMa
H
N
H
N
R
S
R4
19
OH
OMe
H
NO2
>1000
>1000
ni
20
75.5 2.93b
H
N
H
N
R
S
21
OMe
Naphtyl
26
85 0.30
N-Phenylthiourea, N-phenylurea, kojic acid, hydroquinone, arbutin
See Table 1
a
Results are represented as inhibition %, means SE of three independent tests.
Compound 20 was tested at 50 lM; ni, no inhibition.
b
R2
H
N
R'
N
R2
R'
N
R
i
C
+
HN
X
X
R
R4
R4
X = O : 1-14, 18 22 (Tables 1, 2)
19, 20, 21
X = S :
(Table 3)
R; R' : Tables 1, 2, 3
R2; R4 :Tables 2, 3
Scheme 1. Reagents: (i) DMF, NEt3.
R''
N
R'
N
R'
'
R'
N
Cl
i
R
C
O
+
HN
R
O
R'' = Me : 15-17 (Table 1)
Scheme 2. Reagents: (i) DMF, CH2Cl2, NEt3.
inase. Besides, the non-inhibitory property of N-phenylurea re-
vealed the importance of the NHOH moiety for tyrosinase
inhibition.
position of the phenyl ring (4) diminished consequently tyrosinase
activity. Similar results were observed when a –OBu group was
added to the C-4 position (5). For the latter, the increasing length
of hydrocarbon chain drastically affected tyrosinase inhibition.
Addition of –OBn, –NO2, –CF3 or Br at C-4 position of the phenyl
ring resulted in compounds 6 (IC50 = 6.3 lM), 7 (IC50 = 2.6 lM), 10
(IC50 = 4.3 lM), and 11 (IC50 = 2.7 lM), respectively, that exhibited
a potent tyrosinase inhibitory activity, but lower than that of com-
pound 1 or PTU. When R0 position was a methyl, compounds 8 and
12 lost the ability to inhibit tyrosinase activity compared to com-
pounds 7 or 11 (R0 = H). This point is coherent with results
described in Table 1, which showed that an unsubstituted NHOH
m oiety was important for tyrosinase inhibition.
Noteworthy, compounds with hydrophobic electron attractor
groups (CF3, NO2) at C-4 position showed a better efficacy than
compounds with electrodonor groups (OH, OMe, OBu), except for
compounds 6 and 11 (OBn, Br), which exhibited an inhibitory
activity comparable to that of compounds having electroattractor
group at C-4 position. Therefore, it appears that further substitu-
In a next set of experiments, the N-hydroxyurea moiety was
kept unsubstituted, except in two cases for which R0 is a methyl
group (8, 12), and modifications were done at position C-4 of the
phenyl ring (Table 2). Noteworthy, a product with a –OH group
at C-4 position of the phenyl ring should exhibit potent tyrosinase
inhibition by acting as a competitive substrate of tyrosine.25
When a –OH (2) or –NH–CO–NHOH (9) group was attached to
the C-4 position of the phenyl ring, the resulting compounds inhib-
ited tyrosinase activity (respectively, IC50 = 41 and 27 lM), but
were less potent than compound 1. Addition of a hydrophobic
electrodonor –OMe moiety at C-4 position led to compound 3.
The latter showed a better inhibitory effect (IC50 = 32 lM) com-
pared to compound 2 that had a hydrophilic moiety at the same
position. However, addition of a –OMe moiety at both C-4 and
C-2 positions of the phenyl ring (4) prevented the ability of the
compound to inhibit tyrosinase. It seems that substitution on C-2

3610
M. Criton, V. Le Mellay-Hamon / Bioorg. Med. Chem. Lett. 18 (2008) 3607–3610
tions on the phenyl ring should be evaluated for a better under-
standing of their influence on tyrosinase activity. In conclusion,
modification of the C-4 position reduced the ability of compound
1 to inhibit the activity of tyrosinase.
Acknowledgments
The authors are grateful to Dr. Martina Zsely and Dr. Corine Ber-
tolotto for reading this manuscript.
In the last set of experiments, compounds were prepared,
replacing the oxygen of the N-hydroxyurea moiety by a sulfur atom
affording N-hydroxythiourea derivatives (Table 3). Replacement of
the carbonyl oxygen in compound 1 by a sulfur atom resulted in
compound 19 with no inhibitory effect against tyrosinase. Addi-
tionally, methylation of the N–OH moiety and addition of a nitro
group at the C-4 position of the phenyl ring generated compound
20 with no inhibitory potential. On the other hand, replacement
of the phenyl ring by a naphtyl and addition of a –OMe group at
R position (21) improved the inhibitory activity (IC50 = 26 lM)
against tyrosinase compared to compounds 19 and 20.
The effect of the different compounds was next evaluated on
melanin production. To this aim, B16 melanoma cell line was left
untreated or incubated with the different compounds (Table 1–
3). The results revealed that PTU strongly reduced melanin synthe-
sis (58%). In agreement with the results on tyrosinase activity,
compound 1 inhibited melanin synthesis (78%) more efficiently
than kojic acid or arbutin.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.04.079.
References and notes
1. Urabe, K.; Nakayama, J.; Hori, Y. In The Pigmentary System: Physiology and
Pathophysiology; Norlund, J. J., Boissy, R. E., Hearing, V. J., King, R. A., Ortonne, J.
P., Eds.; Oxford University Press: New York, 1998; pp 909–911.
2. Cullen, M. K. In The Pigmentary System: Physiology and Pathophysiology;
Norlund, J. J., Boissy, R. E., Hearing, V. J., King, R. A., Ortonne, J. P., Eds.;
Oxford University Press: New York, 1998; pp 760–766.
3. Sanchez, J. L.; Vazquez, M. Int. J. Dermatol. 1982, 21, 55.
4. Palumbo, A.; d’Ischia, M.; Misuraca, G.; Prota, G. Biochim. Biophys. Acta 1991, 23,
85.
5. Lemic-Stoicevic, L.; Nias, A. H.; Breathnach, A. S. Exp. Dermatol. 1995, 4, 79.
6. Ros, J. R.; Rodriguez-Lopes, J. N.; Garcia-Canovas, F. Biochem. J. 1993, 295, 309.
7. Maeda, K.; Fukuda, M. J. Pharmacol. Exp. Ther. 1996, 276, 765.
8. Battaini, G.; Monzani, E.; Casella, L.; Santagostini, L.; Pagliarin, R. J. Biol. Inorg.
Chem. 2000, 5, 262.
Consistently, most of the compounds that lost inhibitory po-
tential against tyrosinase did not inhibit melanin formation (4,
5, 12, 13, 14, and 19) except for compounds 8 and 20, which
did not inhibit tyrosinase activity but strongly reduced melanin
formation by 82% and 75.5%, respectively. We can hypothesize
that either these two compounds act through a pathway differ-
ent from the inhibition of tyrosinase activity to prevent melano-
genesis or these differences can arise from difference between
mushroom and murine tyrosinase amino acid sequence. On the
other hand, compounds 2, 3, 6, and 22 had an ability to inhibit
tyrosinase activity stronger than kojic acid or hydroquinone but
exhibited low (compound 2) or no inhibitory effect (compounds
3, 6, and 22) on melanin synthesis. This observation could be ex-
plained by their low membrane permeability properties. Finally,
compounds 1, 7, 8, 9, 10, 11, and 21, which were more potent
tyrosinase inhibitors than reference products, were also more
efficient, except for compound 9, in inhibiting melanin produc-
tion compared to kojic acid or arbutin. Noteworthy, the com-
pounds were all assessed for their cytotoxic potential by
measuring viability of B16 cells after incubation with the com-
pounds at different concentrations. The results indicated that
the compounds did not induce cytotoxic effects even at the high-
est concentration (100 lm) (data not shown).
It can be concluded from this whole study that NHOH moi-
ety is important for tyrosinase inhibition. Compound 1 with an
unmodified phenyl ring is the best product we have described
so far. However, further studies including electroattractor
group at C-4 position associated with electron-donating and/
or other electro-attracting groups at other positions should
be done.
Finally among the 22 compounds evaluated in this study, com-
pound 1, inhibiting mushroom tyrosinase with an IC50 of around
0.29 lM and also retaining a substantial potency in cell culture
by reducing pigment synthesis by 78%, may be a promising candi-
date for preclinical drug development for skin hyperpigmentation
application.
9. Ohguchi, K.; Tanaka, T.; Ito, T.; Iinuma, M.; Matsumoto, K.; Akao, Y.; Nozawa, Y.
Biosci. Biotechnol. Biochem. 2003, 67, 1587.
10. Shin, N. H.; Ryu, S. Y.; Choi, E. J.; Kang, S. H.; Chang, I. M.; Min, K. R.; Kin, Y. S.
Biochem. Biophys. Res. Commun. 1998, 243, 801.
11. Kim, Y. M.; Yun, J.; Lee, C. K.; Lee, H.; Min, K. R.; Kim, Y. J. Biol. Chem. 2002, 277,
16340.
12. Curto, E. V.; Kwong, C.; Hermersdorfer, H.; Glatt, H.; Santis, C.; Virador, V.;
Hearing, V. J., Jr.; Dooley, T. P. Biochem. Pharmacol. 1999, 57, 663.
13. Passi, S.; Nazzaro-Porro, M. Br. J. Dermatol. 1981, 104, 659.
14. Gerdemann, C.; Eicken, C.; Krebs, B. Acc. Chem. Res. 2002, 35, 183.
15. Klabunde, T.; Eicken, C.; Sacchettini, J. C.; Krebs, B. Nat. Struct. Biol. 1998, 5,
1084.
16. Farkas, E.; Enyedy, E. A.; Micera, G.; Garribba, E. Polyhedron 2000, 19, 1727.
17. Farkas, E.; Kozma, E.; Petho, M.; Herlihy, K. M.; Micera, G. Polyhedron 1998, 17,
3331.
18. Thatcher, G. R. J. Curr. Top. Med. Chem. 2005, 5, 597.
19. Harmon, R. E.; Dabrowiak, J. C.; Brown, D. J.; Gupta, S. K.; Herbert, M.;
Chitharanjan, D. J. Med. Chem. 1970, 13, 577.
20. Criton, M; Le Mellay, V; Leblond, Y. FR Patent 2880022, 2004.
21. N-Hydroxy-N0-phenylurea (compound 1). To a suspension of hydroxylamine
hydrochloride (17.8 g, 0.248 mol) in dimethylformamide (230 mL) was added
triethylamine (35 mL, 0.249 mol). The reaction mixture was cooled to 0 °C and
phenyl isocyanate (25 mL, 0.225 mol) was added dropwise under N2
atmosphere. The mixture was then allowed to reach room temperature and
stirred for 4 h. The solvent was concentrated and the crude product was
purified by column chromatography using hexane–ethyl acetate (8:2) as
eluent, then recrystallized in ethyl acetate to afford 1 as a white solid (7.02 g,
20.5% yield). Mp (°C): 150–155; 1H NMR (250 MHz, DMSO-d6): d 8.37 (m, 3H,
2NH, –OH), 7.39–7.5 (m, 2H, Ar), 7.2–7.35 (m, 2H, Ar), 6.9 (m, 1H, Ar). MS (ESI
pos): [M+Na]+ m/z = 175.
22. N0-Hydroxy-N-methyl-N-phenylurea (compound 15). To
a suspension of
hydroxylamine hydrochloride (0.5 g, 6.97 mmol) in dimethylformamide
(0.5 mL) and dichloromethane (7 mL) was added triethylamine (7 mmol,
0.98 mL). The reaction mixture was cooled at 0 °C and
a solution of N-
methyl-N-phenylcarbamoyl chloride (1.2 g, 6.97 mmol) in dichloromethane
(5 mL) was added dropwise. The mixture was then allowed to reach room
temperature and stirred for 2 h. The solvent was concentrated and the crude
product was purified by column chromatography using hexane–ethyl acetate
(1:1) to afford 15 (170 mg product, 15 % yield). Mp (°C) 105–110 1H NMR
(250 MHz, DMSO-d6): d 8.20 (m, 2H, NH, –OH), 7.35–7.45 (m, 2H, Ar), 6.9–7.1
(m, 3H, Ar), 3.2 (s, 3H, –CH3). MS (ESI pos): [M+H]+ m/z = 167 and [M+Na]+ m/
z = 189.
23. Dryja, T. P.; O’Neil-Dryja, M.; Pawelek, J. M.; Albert, D. M. Invest. Ophthalmol.
Vis. Sci. 1978, 17, 511.
24. Higashi, Y.; Asanuma, M.; Miyazaki, I.; Ogawa, N. J. Neurochem. 2000, 75, 1771.
25. Nerya, O.; Musa, R.; Khatid, S.; Tamir, S.; Vaya, J. Phytochemistry 2004, 65, 1389.