PEG-immobilization of cardol and soluble polymer-supported synthesis of some cardol-coumarin derivatives: Preliminary evaluation of their inhibitory activity on mushroom tyrosinase

Article abstract of DOI:10.1016/j.bmcl.2008.11.020

In this work, the PEG-immobilization and the liquid phase synthesis of some coumarins derived from cardol are presented. Some preliminary results on their tyrosinase inhibitory activity are also included.

Full text of DOI:10.1016/j.bmcl.2008.11.020

Bioorganic & Medicinal Chemistry Letters 19 (2009) 36–39
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
PEG-immobilization of cardol and soluble polymer-supported synthesis
of some cardol–coumarin derivatives: Preliminary evaluation
of their inhibitory activity on mushroom tyrosinase
b
a
a
a
b
Graziella Tocco a, , Antonella Fais , Gabriele Meli , Michela Begala , Gianni Podda , M. Benedetta Fadda ,
*
Marcella Corda b, Orazio A. Attanasi c, Paolino Filippone c, Stefano Berretta c
a Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, CA, Italy
b Dipartimento di Scienze Applicate ai Biosistemi, Università degli Studi di Cagliari, Cittadella Universitaria di Monserrato, Km 0.700, 09042 Monserrato, CA, Italy
c Istituto di Chimica Organica, Università degli Studi di Urbino ‘‘Carlo Bo”, Via I Maggetti 24, 61029 Urbino, PU, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, the PEG-immobilization and the liquid phase synthesis of some coumarins derived from car-
dol are presented. Some preliminary results on their tyrosinase inhibitory activity are also included.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 29 September 2008
Revised 6 November 2008
Accepted 10 November 2008
Available online 13 November 2008
Keywords:
PEG
Liquid-phase synthesis
Coumarins
Cardol
Tyrosinase
OH
Cardol, 2-methyl cardol and cardanol (Scheme 1) are the main
phenolic components present in the cashew nut shell liquid
(CNSL), an oil obtained as a by-product of the cashew (Anacardium
occidentale L.) nut processing. As renewable raw materials, some of
these could be useful for applications in fine chemical industry.1–4
Chemically, cardols are 5-n-pentadecylresorcinols with satu-
rated, monoolefinic (8), diolefinic (8, 11) or triolefinic (8, 11, 14)
hydrocarbon long side chain.5 Due to their structure, similar to
those of tocopherols, cardols are also known as the most promi-
nent members of the resorcinolic lipids, so called because of their
high lipophilic nature.6 Resorcinolic lipids are of great interest
from many points of view: (a) biotechnological, for the preparation
of biodegradable polyethoxylate surfactants7 or to synthesize soft
materials based on self-assembling aryl glycolipids and used as
nanomaterials (lipid nanotubes, nanofibers, liquid crystals or hy-
dro/organogels as new drug delivery vehicles);8 (b) biopharmaceu-
tical and biomedical, as antimicrobial9–11 and antitumor agents,12
molluscicides13 and prostaglandin synthetase inhibitors.14
OH
OH
H3C
HO
HO
R
R
R
Cardanol
Cardol
2-Methylcardol
R = C15H31
(Cardol 1)
R = C15H29
R = C15H27
R = C15H25
Anacardium Occidentale L.
Scheme 1. Polyphenols isolated from Anacardium occidentale L.
copper-containing enzyme involved in melanin biosynthesis.
Tyrosinase catalyzes, as a polyphenol oxidase, the orto-hydroxyl-
ation of tyrosine to DOPA and the oxidation of L-DOPA to dopaqui-
none, which further polymerizes spontaneously into melanin.
Melanogenesis inhibitors are useful as skin-whitening agents in
the treatment of pigmentation disorders associated with overpro-
duction of melanin, including melasma, solar and senile lentigines,
ocular retinitis pigmentosa19 and Addison’s disease.20
Despite the significant in vitro activity of cardols, especially
those with unsaturated side chain, they, like hydroquinone or
other active compounds,16 can not be used for clinical application,
since they suffer from citoxicity. Furthermore, they could cause
More recently, some naturally occurring compounds such as
coumarins15–17 and cardol18 itself, have gained attention as inter-
esting inhibitors of tyrosinase (EC 1.14.18.1), a multifunctional
* Corresponding author. Tel.: +39 070 675 8711/8551; fax: +39 070 675 8553.
E-mail address: toccog@unica.it (G. Tocco).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.020

G. Tocco et al. / Bioorg. Med. Chem. Lett. 19 (2009) 36–39
37
severe contact dermatitis21 and skin irritation.22 To avoid some of
O
these problems, some b-glycosides of hydroquinone, such as arbu-
O
OH
tin and deoxyarbutin,16 have been synthesized.
R1COCH2COOC2H5
H2SO4 12M
R1
In an attempt to develop an effective and safer tyrosinase inhib-
itor, we have supported the more available cardol 1 on a non-toxic
and versatile polymer such as the poly (ethylene glycol) (PEG),
evaluating, as preliminary results, the activity of the PEG poly-
mer-bound cardol on mushroom tyrosinase.
The selected support was the mono methyl ether of poly (ethyl-
ene glycol) with Mw = 5000 Da, which demonstrated to be easily
soluble in a wide range of solvents, non-toxic, inexpensive, com-
mercially available, easy to functionalize and also resistant to dras-
tic operative conditions.23 Due to these features, PEG chemistry has
shown broad-based application, which may be in large part as-
cribed to the use of PEG-conjugates to deliver drugs, oligonucleo-
tides or enzymes.24
O
C15H31
O
C15H31
6 - 8
5
R1 = -CH3
6
R
1 = -CF3
R1 = -CH2Cl
7
8
Scheme 3. Synthesis of PEG-cardol coumarins 6–8.
OH
OH
O
CH3
The synthetic procedure for the preparation of PEG polymer-
bound cardol25,26 started by anchoring the mono allylated cardol
1 to PEG-mesylate 3 giving the intermediate 4, which was rapidly
deprotected to obtain the final product 5 (Scheme 2).27
Tyrosinase enzyme activity was estimated by measuring the
HO
CH3
10
9
orcinol
rate of oxidation of L
-DOPA to dopaquinone in a modification28 of
O
a previously described method.18 The compound 5 was examined
O
O
O
for its inhibitory activity on mushroom tyrosinase at a concentra-
tion of 0.8 mM, using L-DOPA 0.5 mM as substrate. In these exper-
imental conditions, the PEG-cardol 5 showed an inhibition of
25.37% and an IC50 of 1.52 mM.
CF3
CH3
CH3
CH3
O
O
11
12
A comparison on the activity of compound 5 over cardol 1 was
not possible because of the limited solubility of the last compound.
In fact, in contrast to previous reports,18 cardol 1 started to exhibit
its insolubility at a concentration of 0.2 mM.
Encouraged by these preliminary results and on the basis of our
previous experiences in PEG-supported synthesis,25 we decided to
utilize compound 5 as starting material for the liquid-phase syn-
thesis of some new PEG-cardol coumarins 6–825,26,29 (Scheme 3).
To evaluate the possible influence of the side alkyl chain on
mushroom tyrosinase, similar compounds have been synthe-
sized25,26,30 starting from 5-methylresorcinol 9 (orcinol) instead
of cardol 1 (Scheme 4).
Scheme 4. Synthesis of PEG-orcinol coumarins 11 and 12.
Table 1
Tyrosinase inhibitory activity of PEG-cardol coumarins28
Compounds
Inhibition% (0.8 mM)
[DOPA 0.25 mM] [DOPA 0.5 mM]
IC50(mM)
[DOPA 0.5 mM]
6
11
7
12
21.5
36.19
20
12.3
33
0
1.68
1.73
–
28.5
15.3
3.81
8
0
0
–
Kojic acid
4-Hexylresorcinol
Resorcinol
9.5 Â 10À3
0.98 Â 10À3
1.44
All PEG-coumarins were examined for their tyrosinase inhibi-
tory activity, in the same experimental conditions reported
above.28
*
*
*
*
*
*
The results reported in Table 1 suggest that R groups in the C4
and C5 position of the coumarin skeleton could have a role on the
*
Not tested.
inhibition of the tyrosinase. In fact, 4-methyl-5-pentadecyl-7-O-
PEG-coumarin 6 showed a weaker inhibitory effect than 4,5-di-
methyl-7-O-PEG- coumarin 11. Similarly, 5-pentadecyl-4-trifluo-
romethyl-7-O-PEG-coumarin 7 revealed a decrease in inhibitory
activity when compared with 5-methyl-4-trifluoromethyl-7-O-
PEG-coumarin 12.
O
OH
(a)
HO
C15H31
HO
C15H31
2
1
Furthermore, the presence of a strong electron withdrawing
group in compounds 7 and 12, as well as in 4-chloromethyl-5-
methyl-7-O-PEG-coumarin 8, could be responsible of their reduced
inhibitory activity.
OMs
(b)
3
O
OH
It is remarkable that all the synthesized compounds are com-
pletely soluble in water, suggesting the possibility of performing
the inhibition test without organic solvents. To this intention, the
most active compound 11 was examined in the absence of DMSO,
showing a decrease of about 20% in inhibition, measured in the
usual experimental conditions (DOPA 0.25 mM and 0.5 mM).
Moreover, the effect of PEG on enzymatic activity was excluded. In-
deed, the same experiment performed using PEG as inhibitor, did
not show any decrease of tyrosinase activity. Noteworthy, PEG
proved to be an important synthetic helper, playing a significant
role in the reaction regiochemistry. In fact, compounds 5 and 10
(c)
O
C15H31
O
C15H31
5
4
= MeOPEG- (MW 5000 Da)
Scheme 2. Synthesis of PEG-cardol 5. Reagents and conditions25: (a) allyl bromide,
K2CO3, KI, acetone, reflux, 4 h. (b) Cs2CO3, DMF, 60 °C, overnight. (c) Pd(OAC)2, PPh3,
EtOH, reflux, overnight.

38
G. Tocco et al. / Bioorg. Med. Chem. Lett. 19 (2009) 36–39
12.612 min. Scan:1795 Chan:1 Ion:1774us RIC:153851BC
162
100%
75%
50%
H3C
CH3
190
MCounts
2.5
O
O
HO
14
25%
0%
91
77
147
150
105
51
50
63
133
119
11.760 min. Scan:1657 Chan:1 Ion:1076 us RIC:392682 BC
178
175 m/z
2.0
190
75
100
125
100%
75%
OH CH3
O
O
H3C
1.5
1.0
0.5
0.0
161
13
50%
25%
0%
122
66
150
94
51
50
77
75
105
175
175
133
125
100
150
m/z
minutes
5.0
7.5
10.0
12.5
15.0
Scheme 5. Gas-chromatogram and mass spectra of 4,7-dimethyl-5-hydroxycoumarin 13 and 4,5-dimethyl-7-hydroxy coumarin 14.
20. Pandya, A. G.; Guevara, I. L. Dermatol. Clin. 2000, 18, 91.
21. Diogenes, M. J. M.; de Morais, S. M.; Carvalho, F. F. Int. J. Dermatol. 1995, 34, 72.
22. Stahl, E.; Keller, K.; Blinn, C. Planta Med. 1983, 48, 5.
23. Dickerson, T. J.; Reed, N. N.; Janda, K. D. Chem. Rev. 2002, 102, 3325.
24. Greenwald, R. B. J. Control. Release 2001, 74, 159.
25. Tocco, G.; Begala, M.; Delogu, G.; Meli, G.; Picciau, C.; Podda, G. Synlett 2005, 8,
1296. and references therein.
underwent regioselective cyclization with all the employed b-keto-
esters, just leading to 4,5-dialkyl-7-O-PEG-coumarin derivatives,
independently of the length of the alkyl chain. Conversely, when
we carried out the reaction in solution, under the classical von
Pechmann conditions, it was observed, by means of GC–MS analy-
sis, the formation of the both possible isomers 13 and 14,31 and not
of only one, as previously reported.32–34 (Scheme 5).
26. General Procedure. All PEG samples (Aldrich) were melted in vacuum at 90 °C
for about 45 min before use, to remove any trace of moisture. After reaction,
the crude mixture was concentrated in vacuum to eliminate the solvent, and
than it was added by 6–7 ml of CH2Cl2 to completely dissolve the residue.
The obtained mixture was added to Et2O (50 ml per g of polymer) cooled at
0 °C. The obtained suspension was filtered through a sintered glass filter and
the solid obtained was repeatedly washed on the filter with pure Et2O. All
the samples have been crystallized from isopropyl alcohol, to eventually
eliminate the excess of the polar reagents or the by-products. It is well
known, in fact, that PEGs, as a result of their helical structure, show a strong
propensity to crystallize.25 The yields of PEG-supported compounds were
determined by weight. The indicated yields were for pure products after
crystallization from isopropyl alcohol. Their purity was confirmed by
We can conclude that PEG-conjugates could be valid candidates
as innovative tyrosinase-active compounds, since they are photo-
stable,35 widely soluble in water and in many other solvents and,
last but not least, PEG did not show to affect the activity of an-
chored molecules.
In addition, due to PEG features,23 we think that PEG-cardol and
PEG-cardol coumarins, could lack skin irritation and some other
undesirable side effects.
300 MHz 1H NMR analysis in CDCl3 with
a pre-saturation of the
Finally, PEG confirmed again to be a fundamental tool for the
synthesis of small organic molecules.
methylene signals of the polymeric support at 3.60 ppm. In recording the
NMR spectra, a relaxation delay of 6 s and an acquisition time of 4 s were
used to ensure complete relaxation and accuracy of integration. The
integrals of the signals of the PEG CH2OCH3 fragment at d = 3.30 and 3.36,
were used as internal standards.
References and notes
27. 1-(Allyloxy)-3-O-PEG-5-pentadecylbenzene (4): 1H NMR (300 MHz, CDCl3) ppm:
6.28 (s, 2H), 6.25 (s, 1H), 5.97 (m, 1H, 3J = 5.4 Hz, 3J trans = 17.3 Hz, 3J
cis = 10.5 Hz), 5.34 (d, 1H, 3J = 17.3 Hz), 5.20 (d, 1H, 3J = 10.5 Hz), 4.50 (d, 2H,
3J = 5.4 Hz), 3.36 (s, 3H), 2.45 (t, 2H, 3J = 7.5 Hz), 1.21 (m, 26H), 0.88 (t, 3H, 3J=
1. Tyman, J. H. P. Chem. Soc. Rev. 1979, 8, 499.
2. John, G.; Pillai, C. K. S. Macromol. Rapid. Commun. 1993, 13, 255.
3. John, G.; Pillai, C. K. S. J. Polym. Sci. A Polym. Chem. 1993, 1069, 31.
4. Attanasi, O. A.; Buratti, S.; Filippone, P. Org. Prep. Proced. Ind. 1995, 27, 645.
5. Tyman, J. H. P. In Synthetic and Natural Phenols; Elsevier: Amsterdam, 1996. vol.
52.
7.0 Hz). IR (NaCl) cmÀ1
: m 3100, 2850, 1820, 1650, 1460, 1260, 1050, 820. 3-O-
PEG-5-pentadecylphenol (5): 1H NMR (300 MHz, CDCl3) ppm: 6.27 (s, 1H), 6.22
(s, 2H), 3.33 (s, 3H), 2.43 (t, 2H), 1.21 (m, 26H), 0.83 (t, 3H). IR (NaCl) cmÀ1
: m
6. Kozubek, A.; Tyman, J. H. P. Chem. Rev. 1999, 99, 1.
3550, 3120, 2830, 1610, 1470, 1270.
7. Tyman, J. H. P.; Bruce, I. E. J. Surfactants Deterg. 2003, 6, 291.
8. John, G.; Vemula, K. Soft Matter 2006, 2, 909.
28. The pre-incubation with enzyme consisted of 1.8 ml of a 1/15 M phosphoric
acid buffer solution (pH 6.8), 0.1 ml of an aqueous solution of mushroom
tyrosinase (1000 U/ml, Sigma Chemical Co.) and 0.1 ml of dimethyl sulfoxide
(DMSO) with or without a sample. The mixture was pre-incubated at 25 °C for
9. Himejima, M.; Kubo, I. J. Agric. Food Chem. 1991, 39, 418.
10. Kubo, I.; Muroi, H.; Kubo, A. Bioorg. Med. Chem. 1995, 3, 873.
11. Kubo, J.; Ran Lee, J.; Kubo, I. J. Agric. Food Chem. 1999, 47, 533.
12. Kubo, I.; Ochi, M.; Vieira, P. C.; Komatsu, S. J. Agric. Food Chem. 1993, 41, 1012.
13. Kubo, I.; Komatsu, S.; Ochi, M. J. Agric. Food Chem. 1986, 34, 970.
14. Kubo, I.; Kim, M.; Naya, K.; Komatsu, S.; Yamagiwa, Y.; Ohashi, K.; Sakamoto,
Y.; Hirakawa, S.; Kamikawa, T. Chem. Lett. 1987, 16, 1101.
15. Masamoto, Y.; Murata, Y.; Baba, K.; Shimoishi, Y.; Tada, M.; Takahata, K. Biol.
Pharm. Bull. 2004, 27, 422.
16. Solano, F.; Briganti, S.; Picardo, M.; Ghanem, G. Pigment Cell. Res. 2006, 19, 550.
17. Finn, G. J.; Creaven, B. S.; Egan, D. A. Eur. J. Pharm. Sci. 2005, 26, 16.
18. Kubo, I.; Kinst-Hori, I.; Yokokawa, Y. J. Nat. Prod. 1994, 57, 545.
19. Hartong, D. T.; Berson, E. L.; Dryja, T. P. Lancet 2006, 368, 1795.
10 min. Then, 1 ml of a 1.5 mM L-3,4-dihydroxyphenylalanine (DOPA) solution
was added and the reaction was monitored at 475 nm for 5 min. The
percentage of inhibition of tyrosinase activity was calculated as inhibition
(%) = (A À B)/A Â 100, where A represents the difference in the absorbance of
control sample between an incubation time of 0.5 and 1.0 min, and
B
represents the difference in absorbance of the test sample between an
incubation time of 0.5 and 1.0 min. The activity of mushroom tyrosinase was
determined using
a Varian Cary 50 UV–vis spectrophotometer. DMSO
inhibitory activity, in the same experimental conditions, was evaluated as
10.7%.

G. Tocco et al. / Bioorg. Med. Chem. Lett. 19 (2009) 36–39
39
29. 4-Methyl-5-pentadecyl-7-O-PEG-coumarin (6): 1H NMR (300 MHz, CDCl3) ppm:
cmÀ1
7-O-PEG-coumarin (12): 6.91 (s, 1H), 6.57 (s, 1H), 6.23 (s, 1H), 3.35 (s, 3H), 1.10
(s, 3H). IR (NaCl) cmÀ1
3060, 2835, 1720, 1600, 1550, 1280, 1110.
31. 4,7-Dimethyl-5-hydroxycoumarin (13): 1H NMR (300 MHz, CDCl3) ppm: 6.64 (s,
: m 3060, 2740, 1730, 1665, 1550, 1280, 1100. 5-Methyl-4-trifluoromethyl-
6.69 (s, 1H), 6.45 (s, 1H), 5.98 (s, 1H), 3.40 (s, 3H), 2.54 (s, 3H), 2.43 (t, 2H,
3J = 7.5 Hz), 1.21 (m, 26H), 0.83 (t, 3H, 3J = 7.0 Hz). IR (NaCl) cmÀ1
:
m
3070, 2750,
: m
1760, 1670, 1550, 1280, 1100. 5-pentadecyl-4-trifluoromethyl-7-O-PEG-
coumarin (7): 1H NMR (300 MHz, CDCl3) ppm: 6.71 (s, 1H), 6.47 (s, 1H), 6.23
(s, 1H), 3.32 (s, 3H), 2.42 (t, 2H, 3J= 7.4 Hz), 1.20 (m, 26H), 0.82 (t, 3H, 3J=
1H), 6.61 (s, 1H), 5.99 (s, 1H), 2.78 (s, 3H), 2.27 (s, 3H). IR (NaCl) cmÀ1
: m 3200,
2965, 2880, 1770, 1670, 1456, 1210. 4,5-Dimethyl-7-hydroxycoumarin (14): 1H
7.1 Hz). IR (NaCl) cmÀ1
:
m
3020, 2840, 1770, 1630, 1550, 1280, 1220, 1100. 4-
NMR (300 MHz, CDCl3) ppm: 6.73 (s, 1H), 6.44 (s, 1H), 6.06 (s, 1H), 2.61 (s, 3H),
Chloromethyl-5-pentadecyl-7-O-PEG-coumarin (8): 1H NMR (300 MHz, CDCl3)
2.34 (s, 3H). IR (NaCl) cmÀ1
: m 3195, 2920, 2830, 1770, 1670, 1456, 1210.
ppm: 6.80 (s, 1H), 6.57 (s, 1H), 6.55 (s, 1H), 5.04 (s, 2H), 3.40 (s, 3H), 2.43 (t, 2H,
32. Tianning Diao, J. W.; Sun, W.; Li, Y. Synt. Commun. 2006, 36, 2949.
33. Romanelli, G. P.; Bennardi, D.; Ruiz, D. M.; Baronetti, G.; Thomas, H. G.; Autino,
J. C. Tetrahedron Lett. 2004, 45, 8935.
34. Chakravarti, R. N.; Buu-Hoï, N. P. Bull. Soc. Chim. Fr. 1959, 32, 1498.
35. Tocco, G.; Carbonaro, C. M.; Meli, G.; Podda, G. unpublished results.
3J = 7.5 Hz), 1.21 (m, 26H), 0.83 (t, 3H, 3J= 7.0 Hz). IR (NaCl) cmÀ1
: m 3045, 2800,
1750, 1610, 1550, 1280, 1110.
30. 4,5-Dimethyl-7-O-PEG-coumarin (11): 1H NMR (300 MHz, CDCl3) ppm: 6.71 (s,
1H), 6.48 (s, 1H), 6.01 (s, 1H), 3.33 (s, 3H), 2.56 (s, 3H), 1.06 (s, 3H). IR (NaCl)