S. Genovese, et al.
BioorganicChemistry87(2019)181–190
respectively, compared with control cells that received the solvent ve-
1H), 7.52–7.80 (m, 3H); 13C NMR δ 16.2, 17.3, 18.6, 25.6, 26.2, 39.1,
65.1, 102.0, 106.1, 111.0, 112.4, 119.9, 123.9, 126.3, 131.3, 142.0,
144.8, 152.9, 154.8, 162.3. Anal. Calcd for C20H23ClO3: C, 69.26; H,
6.68; O, 13.84. Found: C, 69.29; H, 6.64; O, 13.87.
hicle alone.
3. Results and discussion
2.2.43. 3-Chloro-4-methyl–7-farnesyloxycoumarin (43)
Previous studies suggested that the presence of prenyl chains linked
to a coumarin ring via ethereal bonds have a deep influence on the
stimulation and or inhibition of melanin biosynthesis in non-tumori-
genic Melan-a cells, an immortalized mouse melanocyte cell line. The
length of these chains are crucial structural determinants to observe a
tanning or a depigmenting effect, in case of compounds with shorter
and longer chains respectively [11]. In the present work it was in-
vestigated and discussed in more details such results using a wider
panel of oxyprenylated chemicals, consisting in 41 natural and semi-
synthetic compounds plus 7-isopentenyloxycoumarin 14 and umbelli-
prenin 17 used as references for the tanning and whitening effects re-
spectively in the above-mentioned murine cell line, in order to study
the impact of the aromatic ring substitutions on the overall melano-
genesis. Samples herein under investigation can be grouped into five
groups, namely benzoic acids (compounds 1–3), benzaldehydes (com-
pounds 4 and 5), cinnamic acid and ferulic acid derivatives (compounds
6–11), anthraquinones (compounds 12 and 13), and coumarins (com-
pounds 14–43), as illustrated in Fig. 1. Among the compounds syn-
thesized, 29, namely 1–18, 22, and 32–40, have been found in nature
as minority phytochemicals of plants mainly belonging to Apiaceae,
Asteraceae, Rhamnaceae, and Rutaceae families [36], as well as com-
ponents of some fungi and marine organisms [37]. The remaining 14,
namely 19–21, 23–30, and 41–43, are of semisynthetic origin. Com-
pounds 20, 33, 34, 40, 42, and 43 are described herein for the first
time. Prenylation and alkylation in general to obtain all chemical
samples have been accomplished following a well validated route pre-
viously reported to provide terpenyl ethers of naturally occurring
phenols [32] as depicted in Scheme 1. Briefly, the substrate was dis-
alkyl bromide or iodide were added. The reaction mixture was let to
react 1 h at 80 °C, poured into icy water and extracted twice with die-
thyl ether to get, after evaporation of the organic solvent to complete
dryness, a raw solid that was further purified by crystallization to
provide the desired compound in pure form and high yield (88%–98%)
without the need for chromatographic separations.
1H NMR δ 1.61 (s, 3H), 1.65 (s, 3H), 1.69 (s, 3H), 1.73 (s, 3H),
2.04–2.16 (m, 8H), 2.54 (s, 3H), 4.48–4.52 (m, 2H), 5.08–5.13 (m, 2H),
5.41–5.44 (m, 1H), 6.31–6.33 (m, 1H), 7.52–7.77 (m, 2H); 13C NMR δ
16.7, 17.8, 18.6, 23.3, 25.7, 26.5, 27.0, 39.6, 39.9, 65.2, 101.9, 106.0,
111.1, 112.4, 118.2, 124.4, 124.8, 126.3, 131.2, 135.0, 141.7, 144.9,
152.9, 154.7, 162.3. Anal. Calcd. for C25H31ClO3: C, 72.36; H, 7.53; O,
11.57. Found: C, 72.32; H, 7.53; O, 11.51.
2.3. Cell culture
Melan-a cells, an immortalized mouse melanocyte cell line, were
obtained from the Wellcome Trust Functional Genomics Cell Bank
(London, UK). Cells were maintained in RPMI 1640 (Lonza, Basel,
Switzerland) supplemented with 10% fetal bovine serum, 50 U/mL
penicillin, 50 U/mL streptomycin (PS Lonza) and 200 nM PMA (phorbol
12-myristate 13-acetate; Sigma). Cells were incubated at 37 °C in a
humidified 5% CO2/air atmosphere. The stock solutions of oxypreny-
lated compounds were prepared in dimethylsulfoxide (DMSO) (1000X)
and were stored at −20 °C until use. The concentration used for the
study was 20 μM, which were freshly prepared for each experiment
with a final DMSO concentration of 0.1%. Controls were always treated
with the same amount of DMSO (0.1%, v/v) as used in the corre-
sponding experiments.
2.4. Cell viability measurements
Non-tumoral murine melanocytes were seeded at 60,000 cells on 6
plate wells and treated for 48 h with the oxyprenylated compound at
20 μM or DMSO. Cells were detached by trypsinization, collected in
phosphate buffer saline and centrifuged at 1500 rpm for 5 min at 4 °C.
Cells pellets were resuspended in the trypan blue solution (0.25%, w/v
in PBS) and counted in a Malassez cell counter under a light micro-
scope. The percentage of cell viability was calculated using the fol-
lowing formula: % cell viability [1 − (blue cells/total cells)] × 100.
All phenols were commercially available, with the only exception of
the starting products for the synthesis of compounds 32–34 and 35–37,
for which preliminary condensation steps between 2,4-dihydrox-
ybenzaldehyde and methyl acetoacetate to yield 3-acetyl-7-hydro-
xycoumarin 44 (Scheme 2), and between the same aromatic aldehyde
and Meldrum’s acid to yield 7-hydroxycoumarin-3-carboxylic acid 45
(Scheme 3) respectively were carried out.
2.5. Melanin content measurements
Non-tumoral murine melanocytes were seeded at 60,000 cells on 6
plate wells and treated for 48 h with the oxyprenylated compound at
the indicated concentration of 20 μM or carrier solvent (DMSO).
5 × 106 cells were centrifuged at 1500 rpm for 5 min at 4 °C. The cell
pellet was washed twice with phosphate buffer saline, transferred in an
Eppendorf vial and centrifuged at 5000g for 5 min at 4 °C. The super-
natant was discarded. 200 μL of H2O and 1 mL of EtOH/Et2O (1/1) were
added to remove opaque contaminants. The mixture was incubated for
15 min at r.t., centrifuged at 5000g for 5 min, and the supernatant
discarded. The precipitate containing melanin was dissolved in 300 μL
of a mixture of 1 M NaOH (aq)/DMSO 9:1 after heating at 80 °C for 1 h.
The absorbance was measured at 405 nm. The melanin content was
expressed as a percentage of control (=100%). UV experiments have
been performed following the method reported by Liebermann and
Hopkins in 2004 [35] and using a UVX radiometer (UVP, Inc., Upland,
CA, USA).
modulate melanin biosynthesis in Melan-a murine cell line. The more
usefulness and better responsiveness of this line as a pharmacological
model in this context respect to other strains, like malignant melano-
cyte cell lines B16F10 and SK-MEL 28 has been well explained in our
previously published manuscript reporting the modulatory effects of
four
naturally
occurring
coumarins,
comprising
7-iso-
pentenyloxycoumarin 14 and umbelliprenin 17, on melanogenesis in
the same non tumoral cell line employed in the present investigation. It
has been preliminarily assayed the impact of oxyprenylated natural and
semisynthetic compounds on proliferation and viability applying a dose
of 20 µM, the same used to carry out tests on the modulation of melanin
biosynthesis and corresponding to the highest solubility of such pro-
ducts into the medium employed to accomplish biological assays. All
synthesized chemicals displayed no significant impact on these two
parameters (data not shown). Thus, all were selected to perform further
experiments. Melanin content was recorded on Melan-a cells exposed to
a concentration of 20 µM of oxyprenylated compounds for 48 h. Results
are reported in Fig. 2. The melanin content of untreated Melan-a cells
2.6. Statistical analysis
Values are the mean
S.E. of three independent experiments, each
carried out in duplicate. Statistical analysis was carried out with
GraphPad using a Student’s t-test for unpaired variables. *, **, and ***
in the figures refer to
P values of < 0.05, < 0.01, p < 0.001
187