Synthesis of Hydroxylated Biphenyl Derivatives Bearing an α,β-Unsaturated Ketone as a Lead Structure for the Development of Drug Candidates against Malignant Melanoma

Article abstract of DOI:10.1002/cmdc.202000709

A small collection of C₂-symmetric hydroxylated biphenyl derivatives featuring an α,β-unsaturated ketone as a lead structure was prepared, and the capacity of these compounds to act as antiproliferative agents against four human malignant melan

Full text of DOI:10.1002/cmdc.202000709

Accepted Article
Title: Synthesis of Hydroxylated Biphenyls Derivatives Bearing an α,β-
Unsaturated Ketone as Lead Structure for the Development of
New Drug Candidates Against Malignant Melanoma
Authors: Maria Antonietta Dettori, Davide Fabbri, Marina Pisano, Carla
Rozzo, and Giovanna Delogu
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000709
Link to VoR: https://doi.org/10.1002/cmdc.202000709
01/2020

10.1002/cmdc.202000709
ChemMedChem
1
1 Synthesis of Hydroxylated Biphenyls Derivatives Bearing an
2 α,β-Unsaturated Ketone as Lead Structure for the Development
3 of New Drug Candidates Against Malignant Melanoma
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Maria Antonietta Dettori,[a] Marina Pisano,[b] Carla Rozzo,[b] Giovanna Delogu,#[a] and Davide
Fabbri*[a]
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[a]
[b]
Dr M.A. Dettori, Dr. D. Fabbri, Dr. G.Delogu.
Consiglio Nazionale Ricerche
Istituto di Chimica Biomolecolare
Traversa La Crucca 3, I – 07100 Sassari, Italy
E-mail: davidegaetano.fabbri@cnr.it
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Dr. M. Pisano, Dr. C. Rozzo
Consiglio Nazionale Ricerche
Istituto di Ricerca Genetica e Biomedica
Traversa La Crucca 3, I – 07100 Sassari, Italy
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#just retired
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Abstract: A small collection of C2-symmetry hydroxylated biphenyls derivatives featured with a α,β-unsaturated ketone as lead structure
was prepared and the capability of such compounds to act as antiproliferative agents against four human malignant melanoma cell lines
was assayed. The prodrug approach was applied in order to improve delivery of compounds into the cell by modulation of the phenolic-
OH protective group. The hydroxylated biphenyl structure bearing an α,β-unsaturated ketone and a phenolic-O-prenylated chain would
facilitated the delivery of the molecule and interactions with the biological targets. Four compounds showed antiproliferative activity
resulting in IC50 value in the range 1.2 - 2.8 µM.
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Introduction
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The search for natural products having antitumoral activity has been rapidly increasing, likely due to the structural diversity
and distinct mechanism of action that natural occurring compounds exert in biological systems.[1] In fact, concomitant inhibition of
multiple pathways required for tumor progression has recently been established as an innovative strategy to improve targeted
therapies in cancer treatment.[2] Cutaneous malignant melanoma (MM) is the most lethal form of skin cancer that arises from
uncontrolled proliferation of melanocytes that are cells producing pigments.[3] Despite noteworthy advances in the field,
heterogeneity and complexity of MM due to several distinct genotypes and phenotypes, make this kind of tumor the most
aggressive form of skin cancer.[4] Therefore, the design of efficient therapies represents a formidable challenge.
It is generally acknowledged that a healthy diet provides beneficial effect as preventive therapy against cancer. Dietary
components of spices and food as curcumin, cinnamon, quercetin, resveratrol, lycopene and epigallocathechins interfere with the
main molecular pathways of melanoma genesis[5] identifying suitable molecular framework on which design new drug candidates.[6,
7] Although curcumin, the main component of Curcuma longa, shows to be extremely safe in animal and humans even at very high
doses, it has not yet been approved as a therapeutic agent due to the poor solubility, stability at physiological level and low
bioavailability (Figure 1).[8] The structure of curcumin, [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione],
consists of a diferuloylmethane framework that exhibits keto-enol tautomerism in solution.[7] The two phenyl unit of curcumin confer
lipophilicity to the molecule whereas the flexible alkylated linker can adopt different conformations contributing to activate effective
interactions with a large number of proteins of pathological relevance.[9] Curcumin contains an α,β-unsaturated Michael acceptor
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pharmacophore that is well recognised in the design of new antimelanoma drugs in virtue of the emerging role of this moiety in
interacting with nucleophiles present in the cancer cells through a Michael addition reaction.[10, 11]
Analogues of curcumin were prepared by independent research groups and the antiproliferative and apoptotic activities
against a wide set of malignancies were assayed identifying lead structures.[7] Most of the curcumin analogues presents
modifications to the flexible unsaturated 1,3-keto-enol moiety which is believed to be responsible for the poor physiological
stability, the poor adsorption and the fast metabolism of the molecule.
Our group prepared an analogue of curcumin, compound 1 (Figure 1) that showed antiproliferative and pro apoptotic
activities against MM and neuroblastoma cells that were ten times stronger than those of curcumin, displaying high selectivity
toward cancer cells.[12, 13] Intracellular concentration of compound 1 reached 600 pmoles/106 cells (about 270 nM) after two hours
after treatment, then degradation of the compound occurred.[13]
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Figure 1. Chemical structures of curcumin, dehydrozingerone and compound 1
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The mechanism underlying the cell growth inhibitory action of compound 1 (compound D6 in the references) was well
investigated.[12-14] In comparison with other analogues of curcumin, the novelty of the molecular framework of compound 1 is a
combination of a hydroxylated biphenyl unit and an α,β-unsaturated carbonyl group with terminal methyl group that reminds the
curcumin structure split in two parts and linked to the aromatic rings. The preparation of the molecular framework relied on a
Claisen-Schmidt condensation between a ketone and an aldehyde under basic conditions.
Hydroxylated biphenyl unit is embedded in many structures of bioactive natural products, some of them of high biological
relevance like ellagitannins and vancomycin, others, structurally less sophisticated, are natural occurring dimers of 4-substituted-
2-methoxy phenols.[15] Hydroxylated biphenyls derivatives have been considered privileged structures due to their unique
pharmacophore able of providing useful ligands for more than one type of receptor.[15] The scaffold shows structural characteristics
such as flexibility combined with partial rigidity, tuneable and fully adaptable in virtue of the presence of certain functional groups.[16]
As result, hydroxylated biphenyl provides an ideal molecular framework for structural modifications in the development of drug
candidates.
Structurally, compound 1, can be also related to two molecules of dehydrozingerone (Figure 1). Dehydrozingerone,
known as feruloylmethane, is isolated from rhizomes of ginger (Zingiber officinale Roscoe), identified as a half structural analogue
of curcumin and one of its degradation compounds at neutral and basic pH conditions.[17] Dehydrozingerone is stable in organic
and aqueous solutions and share many structural and pharmacological features with curcumin.[18] The interesting results achieved
with compound 1 against MM encouraged us to pursue further structural tuning through simple synthetic methods in order to
discover more efficient drug candidates. The substitution in compound 1 of the α,β-unsaturated ketone at 5,5’ positions with an
unsaturated β-diketo enol ester gave comparable results to that of curcumin in term of 50% inhibitory concentration (IC50) against
a set of MM cells and identified the α,β-unsaturated methyl ketone moiety of compound 1 as the most effective Michael acceptor
pharmacophore in the series of the compounds investigated.[19]
With the aim of searching for an effective drug-like Michael acceptor with potential antimelanoma activity, in the present
study, we prepared a collection of derivatives of compound 1. All compounds possess a common α,β-unsaturated ketone moiety
bearing in a hydroxylated biphenyl structure in which the phenolic -OH group was substituted with functionalities able to modify
lipophilicity of the molecule with the aim to increase membrane permeability and bioavailability of the compound. The principal
aim of the work was to prepare compounds that share the same core structure (hydroxylated biphenyl) but differ in patterns of
substituents attached to the core structure. We applied the prodrug approach[20, 21], a successful tool in rational drug design, for
improving bioactivity of compounds by proper transformation of the phenolic -OH group with a functionality that would increase
bioavailability of the molecule or undergone in vivo biotransformation through chemical or enzymatic cleavage, thus modulating
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pharmacokinetic properties and/or favouring the delivery of the active compound with a higher yield. Further, all compounds were
assayed in vitro on a set of human MM cell lines and their IC50 determined.
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Results and discussion
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Chemistry
We started from the experimental evidence that compound 1, a tetra-OMe biphenyl (D6 in references 12-14) showed
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antiproliferative and apoptotic activity in malignant melanoma cells with negligible effect on normal cells.[13] A collection of eighteen
derivatives of 1, compounds 2-19, were prepared through straightforward synthetic procedures. Compounds 2-13 possess a
hydroxylated biphenyl bearing an α,β-unsaturated ketone as lead structure where phenolic –OH group was transformed with
aliphatic group/chain with the aim to improve antimelanoma activity in comparison with that assayed in compound 1 (Figure 2).
Compounds 14-19 are the corresponding monomers of hydroxylated biphenyls derivatives 1, 4, 5 and 11-13. We applied the
prodrug approach[20, 21] and tuned the structure of compound 1 in such a way that the hydroxyl groups at the 2,2’ positions were
transformed with a hydrolisable carrier (carrier-linker prodrug) as for compounds 4, 6, 7, 9 and monomer 18 or with a functional
group that could provide synergistic action after hydrolysis (mutual prodrugs) as for compounds 8.[22] Biphenols 2, 3 and 5 and
monomer 19 were prepared as representative of compound 1 with increased hydrophilicity and biphenyl 10 as the most lipophilic
compound. Concerning oxo-prenylated ethers 11-13 and 15-17 we though to increase bioavailability and selectivity of the
compounds because oxo-prenylated phenols are able to interact with different and selected cell receptors and signal transductors
(ex. Ras proteins) accounting for their ability to modulate key metabolic processes in pathological disorders.[23]
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Figure 2. Chemical structures of compounds 2-19.
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Similarly, as in 1, the presence of a C2-symmetry axis in compounds 2-13 allows having the two aromatic rings
indistinguishable, this structural feature provides an increase in reaction selectivity. The scarce water solubility is one hallmark of
compound 1 when it was diluted in physiological medium[12] thus, free phenolic –OH groups at 2,2ʹ-positions of compound 1 were
obtained by Claisen-Schmidt reaction with dehydrodivanillin and acetone in the presence of Li(OH) to give compound 2 in almost
80% yield.[7] The compound was the starting material for the synthesis of compounds 4 and 6-12 (Scheme 1).
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Scheme 1. Reagents and conditions: (a) compound 3: BBr3 in CH2Cl2, -60 °C for 1h; (b) compound 6: methyl chloroformate, Et3N at rt in CH2Cl2 for 1 h; (c)
compound 7: glutaric anhydride, DMAP at reflux in THF for 12 h; (d) compound 8: octanoyl chloride, Et3N at rt in CH2Cl2 for 4 h; (e) compound 9: ethyl-3-
(chloroformyl)propionate, Et3N at rt in CH2Cl2 for 96 h. (f) compound 10: 1-bromooctadecane, K2CO3, 18-crown-6 ether in CH3CN, reflux for 12 h; (g) compound
11: allyl bromide, K2CO3 in acetone, reflux for 12 h; (h) compound 12: 3,3'-dimethylallyl bromide, K2CO3 in acetone, reflux for 12 h; (i) compound 4: 2,3,4,6-
tetra-O-acetyl-α-D-glucopyranosyl bromide, Ag2CO3 at rt in pyridine for 12 h, then, after purification of the product, CH3OH/CH3ONa at rt for 10'.
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Catechol units were achieved from compound 2 with a large excess of equivalents of demethylating reagent at -60 °C
for 1 h giving compound 3 in 89% yield. The browning of compound 3 after few days from the preparation was likely a consequence
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of the easy oxidation of the compound, therefore storage of compound 3 under nitrogen atmosphere was mandatory. While
compound 2, C2-dimer of dehydrozingerone, showed scarce solubility in water and in physiological solution at concentrations >
2mM, compounds 3 and 4 were completely soluble in water up to 30 mM, accordingly to their LogP (Table 1).
Table 1. Values of the logarithm of the partition coefficient of compounds 1-19 for n-octanol/water (LogP), estimated by ChemBioDraw13 Ultra 13.
Compounds
LogP
Compounds
LogP
Compounds
LogP
Compounds
LogP
1
2
3
4
5
2.70
2.17
1.65
-1.51
-2.22
6
7
3.24
2.29
7.60
3.64
>8.50
11
12
13
14
15
4.08
5.17
8.36
1.53
2.22
16
17
18
19
2.76
3.87
8
-0.57
-0.93
9
10
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Then, we looked upon for molecular variations in developing a series of derivatives of 1 having different functional
group/unit linked to the phenolic -OH group with the aim to improve bioavailability. First of all, the choice fell on easily hydrolysable
leaving groups as acetals, carbonates and esters. Different hydrolysis rate would be expected for these classes of compounds
releasing compound 2 at different compartments of the cell.[24]
Phenolic -OH groups of compound 2 were protected with a glycosylated unit starting from 2,3,4,6-tetra-O-acetyl-α-D-
glucopyranosyl bromide and silver carbonate under basic conditions and further hydrolysis of acetyl groups following a known
procedure that allowed to achieve compound 4 as β-anomer selectively.[25]
Methyl chloroformate with diol 2 in the presence of trimethylamine as base, gave dicarbonate 6 in 91% yield. With the
aim to introduce an ester leaving group in an aliphatic chain with different lipophilicity and bioactivity, compounds 7-9 were
prepared. Compound 7, achieved by reaction of compound 2 with glutaric anhydride under basic conditions possesses, for each
aromatic ring, an ester and a carboxylic acid functionality between three-methylene carbon chain. Seven-methylene carbon chain
represents the octanoic acid portion of diester 8 whereas compound 9, prepared by reaction with ethyl-3-(chloroformyl)propionate,
possesses two-methylene carbon chain that links two ester functionalities. Although compounds 7-9 contains ester groups
embedded onto the same hydroxylated biphenyl-α,β-unsaturated ketone moiety, compound 7 possesses a terminal carboxylic
groups for each aromatic ring that make the molecule less lipophilic (LogP 2.29) than that of ester 9 (LogP 3.64) whereas ester 8
having a medium-chain fatty ester, is highly lipophilic (LogP 7.60). According to the pro-drug approach, compound 8, after ester
hydrolysis, should provide biphenyl 2 and octanoic acid (i.e. caprylic acid), two molecules with remarkably reduced lipophilicity in
comparison to 8.
In compound 9, the phenolic -OH is protected by a small chain featuring two ester functionalities that would facilitate the
delivery of the molecule through the lipophilic cell membrane. Compound 9 can also exert the role of linker due to the easy
hydrolyse of the ethyl ester group by esterases[26] producing a carboxylic acid group at the end of each small aliphatic chain. It
would not be ruled out the expected amphiphilic properties of compound 9 when hydrolysis is applied. In the attempt to achieve
another analogues of compound 1 with amphiphilic properties, a glycosylated unit was introduced at the end of the α,β-unsaturated
ketone chain. Claisen-Schmidt condensation of OMe-dehydrodivanillin with O-per-acetilated-β-C-glucopyranosyl ketone in the
presence of pyrrolidine as base and further deacetylation, gave compound 5 in 73% overall yield (Scheme 2).
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Scheme 2.Reagents and conditions: (a) OMe-dehydrodivanillin: from dehydrodivanillin, ref. 26; (b) compound 5: per-O-acetylated-β-C-glucopyranosyl ketone,
pyrrolidine at rt in CH2Cl2, 48 h, then, after purification of the product, CH3OH/CH3ONa at rt for 10'; (c) compound 11: allyl bromide, K2CO3 in acetone, reflux,
12 h, then, after purification of the product, 1N NaOH solution in acetone at rt for 12 h. (d) compound 13: geranyl bromide, K2CO3 in acetone, reflux, 12 h,
then, after purification of the product, 1N NaOH solution in acetone at rt for 12 h.
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The presence of two glycosylated units at the end of the α,β-unsaturated ketone chain provided in compound 5 a
significant increase of hydrophilicity in comparison with that evaluated for compounds 4 bearing the glycosylated units at the
phenolic –OH groups (Table 1).
The pro-drug approach was also applied in the synthesis of compound 9 in order to improve delivery or selectivity of the
molecule in physiological environment under in vivo biotransformation through chemical or enzymatic cleavage[20, 21]. The highest
lipophilicity was achieved with compound 10 prepared at room temperature by Williamsons ether reaction of 2 with 1-
bromooctadecane in the presence of 18-crown-6 ether as catalyst.
Different prenylated chains units were introduced at the phenolic -OH group of compound 2 by Williamson ether reaction,
under basic conditions and in the presence of the corresponding prenylated bromide, as organo halide. Compounds 11 and 12
which contains an allylic and 3,3ʹ-dimethyl allyllic ether chain, respectively, were prepared in a range of 60-65% yield starting from
2 (Scheme 1). An alternative procedure was applied to compounds 11 and 13 that entailed Williamson ether reaction of
dehydrodivanillin with the corresponding prenylated bromide, then the aldehyde group of the product was transformed in a α,β-
unsaturated methyl ketone moiety by Claisen-Schmidt condensation in the presence of acetone under basic conditions (Scheme
2). Monomers 14-17 were prepared with the same synthetic procedures described in literature for these compounds that entailed
protection of the phenolic -OH group of dehydrozingerone with the corresponding organohalide under basic conditions.[27, 28]
According to the reaction stoichiometry, monomers 18 and 19 were prepared with the same procedures applied for the synthesis
of the corresponding dimers 4 and 5, respectively (Scheme 3).
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Scheme 3. compound 18: (a) 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide, Ag2CO3 at rt in pyridine for 18 h, then, after purification of the product,
CH3OH/CH3ONa solution at rt for 10'; compound 19: (b) per-O-acetylated-β-C-glucopyranosyl ketone, pyrrolidine at rt in CH2Cl2, 72 h, then, after purification
of the product, MeOH/MeONa at rt for 10'
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Remarkable different lipophilicity was estimated between the series of the monomer and dimers, in the latter, the
difference was more evident as the number of carbon atoms in the prenylated chain increases.
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Biological evaluation
We explored the bioactivity changes in compounds 2-19 induced by transformation of the functional phenolic -OH group
underlying cell growth arrest in four MM cells lines, labelled LCP, LCM, CN and M14. We chose these four cell lines from a larger
panel, for the following reasons: CN was one of the cell lines already demonstrated to be sensitive to compound 1[12] LCP and
LCM were a couple of cell lines deriving from different stage tumor lesions of the same melanoma patient, being LCP derived
from the primitive lesion and LCM from a lymph node metastasis; M14 was a commercially available melanoma cell lines and can
be considered as a melanoma reference in vitro model.[29] In our previous work[12] compound 1 showed an average IC50 of 1.8 ±
0.9 µM, average calculated on the basis of results obtained from 72h proliferation assays on five cell lines, including CN. Here, in
a preliminary experiment, the antiproliferative activity of compounds 2-19 was calculated as IC50 value on the basis of proliferation
assays performed treating cells up to 72h. Subsequently, compounds with IC50 value below 7.0 µM on all the cells lines
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(compounds 6-9 and 11, 12) were tested after 24h treatments (Table 2). Moreover, our most active compounds 11 and 12 were
also tested on BJ normal fibroblast cell line, as a non-tumor cells control, in order to assess their selectivity.
With the aim to improve the antiproliferative activity of compound 1 (IC50 1.0-2.7 µM)[12], we modified its physical-chemical
properties by introducing at the 2,2ʹ-positions of the biphenyl structure different groups/chains by tuning lipophilicity, leaving group
and ability to undergone chemical/enzymatic hydrolysis in the cell. Moreover, it should be kept in mind the crucial role that
lipophilicity plays in designing oncological drugs because facilitates cell membrane access of the compound in the required critical
concentration.[30]
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Table 2. Cytotoxicity (IC50 in µM) of compounds 2-19. IC50 values represent mean value (±SD) of three independent measurements each performed in
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triplicate. The cell lines LCP, LCM, CN, M14 are primary human MM cell lines, as described in detail in the experimental section.
LCP
LCM
CN
M14
Compounds
IC50
IC50
IC50
IC50
24 h
72 h
24 h
72 h
24 h
72 h
24 h
72 h
2
3
n.d
6.0 ± 0.8
n.d.
34 ± 3.2
n.d.
27 ± 1.8
n.d.
56 ± 5.1
n.d.
n.d.
79 ± 8.3
>100
n.d.
n.d.
>100
>100
n.d.
n.d.
55 ± 4.5
>100
n.d.
n.d.
38 ± 2.6
>100
4
5
n.d.
>100
n.d
>100
n.d.
>100
n.d.
>100
6
7.5 ± 1.1
>10
1.3 ± 0.7
4.1 ± 0.8
2.1 ± 0.5
4.0 ± 0.7
n.s.
>10
1.6 ± 0.4
3.7 ± 0.7
1.9 ± 0.3
2.2 ± 0.3
n.s.
>10
1.8 ± 0.2
3.7 ± 0.8
1.8 ± 0.2
3.5 ± 0.4
n.s.
3.6 ± 0.1
>10
2.5 ± 0.4
6.2 ± 1.1
1.6 ± 0.9
3.6 ± 0.9
n.s.
7
>10
>10
8
>10
7.0 ± 1.5
7.5 ± 1.0
n.s.
6.4 ± 1.4
>10
4.4 ± 1.4
4.6 ± 1.2
n.s.
9
>10
10
11
12
13
14
15
16
17
18
n.s.
n.s.
6.6 ± 1.0
6.7 ± 1.3
n.d.
1.6 ± 0.1
2.4 ± 0.6
15 ± 3.2
>100
7.9 ± 1.8
8.8 ± 2.5
n.d.
2.4 ± 0.6
2.8 ± 0.8
8.5 ± 1.1
>100
7.9 ± 1.5
5.6 ± 1.3
n.d
1.6 ± 0.2
1.6 ± 0.1
7.8 ± 2.7
>100
2.4 ± 0.9
6.5 ± 1.0
n.d.
1.2 ± 0.2
1.2 ± 0.1
5.5 ± 1.7
>100
n.d.
n.d.
n.d.
n.d.
n.d.
36 ± 4.2
37 ± 5.0
29 ± 2.7
>100
n.d.
32 ± 3.8
39 ± 3.5
43 ± 3.8
>100
n.d.
32 ± 3.7
32 ± 4.1
31 ± 2.9
>100
n.d.
34 ± 2.6
24 ± 2.9
24 ± 2.1
>100
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
19
n.d.
>100
n.d.
>100
n.d.
>100
n.d.
>100
213
n.s. = not soluble
n.d.= not determined
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C2-symmetry is a powerful tool in organic synthesis because when this element is present in a substrate, a control of
the production of isomers occurs improving the selectivity of the reaction. The presence of a C2-symmetry axis in compounds 2-
13 is a useful structural element also from a biological point of view because identical interspecific interactions can be activated
between each symmetrical portion of the molecule and target proteins, thus providing an increased selectivity and control in the
interaction molecule-protein.
The hydrophilic compounds 2 and 3 featured with two and four free phenolic –OH groups, respectively, affected
melanoma cells after 72h with different antiproliferative activity (Table 2). Compound 2, with guaiacyl units and similar to compound
1, was more effective than 3 against LCP, LCM and CN cell lines with IC50 ranging from 6 ± 0.8 to 34 ± 3.2 µM. Previously, we
found that compound 2 was effective breaking-chain activator in bulk lipid autoxidation and showed strong cytotoxicity against rat
pheochromocytoma (PC12) cells, a slow-growing neuroendocrine tumor cell line.[31, 27]
Glycosylation of compound 2 was detrimental for antiproliferative activity on all melanoma cell lines. Derivatives 4 and 5
with a O-glycoside unit linked at the phenolic -OH group and a C-glycoside unit at the end of the aliphatic chain, respectively, both
gave value of IC50 >100 µM after 72 h of treatment. Likely, the high hydrophilicity of 4 and 5 hindered complete bioavailability of
the compounds into the cell membrane. Same results were obtained with the corresponding monomers 18 and 19 evidencing the
key role of lipophilicity of the molecule in the delivery through the cell membrane.
Enzyme-catalysed hydrolysis of the methyl carbonate ester 6 would provide release of the biphenyl portion in the cell as
experienced when bioactive methyl phenols carbonates were screened on M14 cell line, as result, higher antiproliferative activity
was observed in comparison with the parent phenol.[6] In our work, compound 6 was more effective in the growth inhibition of M14
cell line (IC50 = 3.6 ± 0.1 µM) than in the other cell lines after 24h. As proof of the hydrolysis of carbonate functionality, compound
6 was more effective against LCP, LCM and CN cell lines after 72 h of treatment, reaching IC50 values below 2 µM. It is likely that
during this time, compound 6 or the corresponding hydrolysed forms reached more sensitive cell compartments in comparison to
compound 2 that is the complete hydrolysed form of 6. A similar trend was observed when cells were treated with compounds
with hydrolysable esters (i.e. compounds 7-9), the antiproliferative effect improved on all cell lines after 72h giving IC50 values in
the range of 1.6 ± 0.9 and 6.2 ± 1.1 µM. In comparison with IC50 of compound 2 achieved after 72h, it is reasonable to suppose a
synergistic effect of the hydrolysable chain of compounds 7-9 with the hydroxylated biphenyl portion providing an increase of the
antiproliferative activity.
Although IC50 value is strictly dependent on the cell type, significant antiproliferative activity was observed after 24h of
treatment of LCM, CN and M14 cell lines with compound 8, the activity improved after 72h on all melanoma cell lines. A remarkable
increased lipophilicity was estimated for compound 8 (Table 1), likely crucial in exerting an efficient delivery of the compound
through the lipophilic cell membrane. Differences in the invasiveness of tumor cells could derive, among different advantageous
features acquired during tumor transformation, from the activity of secreted and membrane-associated enzymes.[24] Noteworthy,
an octanonyl ester function is present in compound 8. It is well recognised the role of medium chain fatty acids, in particular
octanoic acid (i.e. caprylic acid)[22], in exerting antiproliferative activity against skin cancer in vitro[32]and, in vivo, in activating
endogenous host peptides targeted to enhance intestinal epithelial immunological barrier.[33] In this work we did not investigated
the real role of the octanoyl chain, but we observed the improvement of the antiproliferative activity of compound 8 in comparison
with esters 6, 7 and 9 after 72 h of treatment.
An excessive increase in lipophilicity was detrimental to bioavailability. Data achieved from compound 10 having a too
long lipophilic aliphatic ether chain, were excluded from Table 1 since not reliable due to the scarce solubility of the molecule in
water even at low concentrations.
We assayed compounds 11-13, having a small, hindered and long O-prenylated chain, respectively, because there is
convincing evidence that natural and synthetic oxyprenylated phenylpropanoids assume an important role in inhibiting some
malignant cells.[34] Several mechanisms of action have been attributed to prenylated aromatic phenols.[23, 35].Generally, it was
demonstrated that oxyprenylated phenols are able to interact with different and selected cell receptors and signal transductors
(ex. Ras proteins) accounting for their ability to modulate key metabolic processes in pathological disorders. Likely, their effect is
immediately exerted on membrane cell due to the high affinity with the phospholipidic portion, favouring bioavailability of the
compound.[36] In breast cancer, some oxyprenylated ferulic acids with 3,3ʹ-dimethyl allyl and geranyl moiety at the phenolic -OH
group, were successfully assayed for their binding affinities to MT1 melatonin receptors[37] and the antiproliferative and
antimigratory properties were detected at µM concentration levels. In our work, allyl and 3,3’-dimethyl allyl O-prenylated
compounds, 11 and 12 respectively, showed comparable antiproliferative activity assessed after 72h in a range between 1.2 ±
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0.1 and 2.8 ± 0.8 µM and in higher level in comparison with that showed by compound 13 bearing a O-geranyl chain (IC50 5.5 ±
1.7 – 15 ± 3.2 µM). It would not be ruled out that the higher lipophilicity of compound 13 (LogP 8.36) in comparison to compounds
11 and 12 (LogP 4.08 and 5.17, respectively) could likely facilitate membrane penetration but hinder diffusion of compound 13
inside the cell. After 24h, compounds 11 and 12 showed interesting antiproliferative activity on all MM cell lines although M14 cell
line appeared more sensitive to compound 11 even after 24h treatment (IC50 2.4 ± 0.9). In a lower order of magnitude,
antiproliferative activity was observed after 72h with the corresponding monomers 15-17 accounting for IC50, homogeneously
distributed on all MM cells lines, between 24 ± 2.1 – 43 ± 3.8 µM for all three compounds. When the phenolic -OH group was
protected with a methyl group (i.e. compound 14), the activity dropped significantly. Overall, the last results confirmed the
beneficial influence that a small phenolic-O-prenylated chain exerts in an α,β-unsaturated Michael acceptor in enhancing
antiproliferative activity of the molecule. A comparison of IC50 between monomers 15-17 and dimers 11-13 evidenced the key role
of the hydroxylated biphenyl core in enhancing antiproliferative activity and in modulating the physical-chemical properties of the
molecule.
Interestingly, compounds 8, 11 and 12 showed comparable antiproliferative activity after 72h on all MM cells lines even
though differences between O-prenylated compound 11 and ester 8 were observed in LCP and M14 cell lines after 24h of
treatment. The effect could be due to the different rate of hydrolysis of the protecting group or different selectivity when the
compounds are in the presence of cell specific enzymes or membrane transporters. Moreover, the activity of compounds 11 and
12 was much lower on BJ fibroblasts, the healthy, non-tumor cell line used as control. BJ cells were given the same treatments
as MM cells, and showed to be much less affected by them. Indeed, compounds 11 and 12 showed significantly higher IC50 values
after 72h of treatments (6.4±0.2 M and 6.0±0.6M, respectively) when compared to the mean IC50 value of all MM cell lines
(1.7±0.5M for 11 and 2.0± 0.7M for 12) (p = 0.000024 and p = 0.0006299 respectively). The cytotoxicity ratio between cancer
and healthy cells was 0.27 for 11 and 0.33 for 12. These last evidences suggest a selectivity of antiproliferative activity against
tumor cells for these two compounds. Such a difference should be related to the higher cell division activity of tumor cells, and
might be exploited in future anticancer therapies designing. Selective antitumor activity was one of the major features of compound
1 [12,13], and the present results show that it has been retained by compounds 11 and 12.
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Conclusions
We have prepared a small collection of C2-symmetry hydroxylated biphenyls derivatives bearing a α,β-unsaturated
ketone, compounds 2-13, structurally related to compound 1, known for selective and effective antiproliferative activity on MM
cells.
The synthesis of compounds 2-19 was carried out in order to improve delivery of compounds into the cell by modulation
of the phenolic –OH protective group. The prodrug approach was applied in the synthesis of biphenyls 4, 6-9 and monomer 18.
Different functional groups were introduced by straightforward methods giving ethers, esters, carbonate and acetal derivatives
that influenced the physical-chemical properties of the molecule, mainly its lipophilicity and the capacity of hydrolysis into the cell
or the selectivity toward tumor targets. By tuning the physical-chemical properties, we were able to identify the core molecular
scaffold characterized by an hydroxylated biphenyl core bearing an α,β-unsaturated methyl ketone protected at the phenolic -OH
group with a small O-prenylated chain, compounds 11 and 12. Although the water solubility and the selective antiproliferative
activity on MM cells of these two compounds showed to be comparable to those of compound 1, this work evidenced the
importance of a phenolic-O-prenylated chain in the structure of compound 1, likely crucial for delivery of the molecule and
interactions with the biological targets. Further studies devoted to shed light in the mechanism of compound 11 and 12 will be
object of a next work.
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Experimental
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Material and general remarks
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Unless otherwise noted, starting materials and reagents were obtained from commercial suppliers and were used
without further purification. Melting points were determined on a Büchi 530 apparatus and are uncorrected. All 1H NMR and
13C NMR spectra were recorded in CDCl3 (if not otherwise indicated) solution with a Varian VXR 5000 spectrometer at 399.94
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MHz and 75.42 MHz respectively. Chemical shifts are given in ppm (); multiplicities are indicated by s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet) or dd (double of doublets). Elemental analyses were performed using an elemental analyser
Perkin-Elmer model 240 C. Acetone was freshly distilled from CaCl2. Flash chromatography was carried out with silica gel 60
(230-400 mesh, Kiesgel, EM Reagents) eluting with appropriate solution in the stated v:v proportions. Analytical thin-layer
chromatography (TLC) was performed with 0.25 mm thick silica gel plates (PolygramSil G/UV254, Macherey-Nagel). All
reactions were monitored by TLC performed on 0.2 mm thick silica gel plates (60 F254 Merck). The purity of all new
compounds was judged to be >98% by 1H NMR and 13C NMR spectral determination.
Compounds 2 and 14 were prepared according to Marchiani et al. [7] whereas compounds 15-17 were prepared as described
by Tatsuzaki et al.[28] Dehydrodivanillin and OMe-dehydrodivanillin (i.e.2,2ʹ,3,3ʹ-tetramethoxy-5,5ʹ-diformyl-1,1ʹ-bipheny (23),
were obtained as described by Pisano et al.[12] whereas per-O-acetylated β-C-glucopyranosyl ketone was prepared following
the procedure described by Llantén et al.[25].
Lipophilicity of compounds 1-19 was estimated by ChemBioDraw Ultra 13.0 software using the logarithm of the partition
coefficient for n-octanol/water (LogP) and listed in Table 1.
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Chemical synthesis
(3E,3'E)-4,4'-(5,5',6,6'-tetrahydroxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one) (3)
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To a solution of 2 (0.6 g, 1.57 mmol) in dichloromethane (20 mL) at -60 °C under nitrogen was added boron tribromide
(1.71 g, 6.95 mmol) dropwise. The solution was stirred at -60 °C for 1h, washed with water (100 mL) and extracted with ethyl
acetate (2 x 20 mL). The organic solution was dried over sodium sulphate, rotoevaporated and washed with dichloromethane
(2 x 10 mL) to give 3 as a yellow solid. (0.47 g, 89%): mp = 220-222°C; 1H NMR (CD3OD)  2.34 (s, 6H), 4.90 (bs, 4H), 6.58
(d, J = 16.4 Hz, 2H), 7.07 (d, J = 2.0 Hz, Ar, 2H), 7.11 (d, J = 2 Hz, Ar, 2H), 7.56 (d, J = 16.4 Hz, 2H); 13C NMR (CD3OD) 
25.61, 112.16, 123.64, 124.41, 125.89, 125.92, 145.45, 146.19, 146.35, 200.15. Anal. Calcd. for C20H18O6: C, 67.79; H, 5.12;
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Found: C, 67.83; H, 5.16.
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(3E,3'E)-4,4'-(5,5'-dimethoxy-6,6'-bis(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-
[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one) (4)
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Compound 22 (1 g, 0.96 mmol) was stirred in sodium methoxide/methanol solution (0.02 g, 0.38 mmol in 10 ml) for
10 min. The reaction mixture was neutralized using a Dowex Marathon C (H+ form), filtrated and concentrated in vacuo to
obtain 4 as a yellow solid. (0.61 g, 90%): mp = 125-126°C; [α]D20 10.7 (c 0.2, MeOH); 1H NMR (CD3OD)  2.36 (s, 6H), 3.10-
3.17 (series of m, 4H), 3.23-3.34 (series of m, 4H), 3.61 (dd, J = 4.8, 11.6 Hz, 2H), 3.72 (dd, J = 2.0, 11.6 Hz, 2H), 3.93 (s,
6H), 5.23 (bs, 2H), 6.78 (d, J = 16.4 Hz, 2H), 7.29 (s, Ar, 2H), 7.47 (bs, Ar, 2H), 7.65 (d, J = 16.4 Hz, 2H); 13C NMR  (CD3OD)
26.02, 55.42, 60.94, 69.91, 74.19, 76.28, 76.93, 101.55, 111.22, 125.54, 125.86, 129.91, 144.38, 144.54, 152.30, 163.12,
200.18. Anal. Calcd. for C34H42O16: C, 57.79; H, 5.99; Found: C, 57.80; H, 5.96.
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(5,5',6,6'-tetramethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)but-3-en-2-one) (5)
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Compound 24 (0.17 g, 0.15 mmol) was stirred in sodium methoxide/methanol solution (0.003 g, 0.06 mmol in 10 ml)
for 10 min. The reaction mixture was neutralized using a Dowex Marathon C (H+ form), filtrated and concentrated in vacuo to
obtain 5 as a brown solid. (0.11 g, 95%): mp = 140-141°C; [α]D20 19.1 (c 0.25, MeOH); 1H NMR (CD3OD)  2.88 (dd, J = 9.2,
16.4 Hz, 2H), 3.09-3.18 (series of m, 4H), 3.22 (m, 2H), 3.36 (m, 2H), 3.58-3.64 (series of m, 10H), 3.74 (m, 4H), 3.95 (s, 6H),
6.85 (d, J = 16.0 Hz, 2H), 7.10 (s, Ar, 2H), 7.32 (s, Ar, 2H), 7.62 (d, J = 16.0 Hz, 2H); 13C NMR  (CD3OD) 42.96, 55.16, 59.80,
This article is protected by copyright. All rights reserved.

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61.31, 70.21, 73.72, 76.08, 78.25, 80.15, 111.12, 123.93, 125.41, 130.16, 132.41, 143.36, 148.92, 153.01, 199.80.Anal. Calcd.
for C36H46O16: C, 58.85; H, 6.31; Found: C, 58.90; H, 6.37.
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3,3'-dimethoxy-5,5'-bis((E)-3-oxobut-1-en-1-yl)-[1,1'-biphenyl]-2,2'-diyl dimethyl dicarbonate (6)
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To a solution of 2 (0.9 g, 2.35 mmol) and triethylamine (0.51 g, 5.04 mmol) in dry dichloromethane (10 mL) methyl
chloroformate (0.55 g, 5.92 mmol) was added dropwise, at room temperature under nitrogen. The solution was stirred at room
temperature for 1 h, washed with water (100 mL) and extracted with dichloromethane (2 x 20 mL). The crude was dried over
sodium sulphate, rotoevaporated and washed with diethyl ether to give 6 as a white solid (1.01 g, 91%): mp = 182-183°C; 1H
NMR  2.37 (s, 6H), 3.78 (s, 6H), 3.92 (s, 6H), 6.64 (d, J = 16.0 Hz, 2H), 7.06 (d, J = 2.0 Hz, Ar, 2H), 7.16 (d, J = 2.0 Hz, Ar,
2H), 7.45 (d, J = 16.0 Hz, 2H); 13C NMR  27.62, 55.66, 56.25, 111.14, 123.07, 127.81, 130.75, 133.03, 139.47, 142.16,
151.97, 152.90, 197.92. Anal. Calcd. for C26H26O10: C, 62.65; H, 5.26; Found: C, 62.70; H, 5.24.
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5,5'-((3,3'-dimethoxy-5,5'-bis((E)-3-oxobut-1-en-1-yl)-[1,1'-biphenyl]-2,2'-diyl)bis(oxy))bis(5-oxopentanoic acid) (7)
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To a solution of 2 (2 g, 5.23 mmol) and glutaric anhydride (1.32 g, 11.57 mmol) in tetrahydrofuran (100 mL) was
added N, N-dimethylaminopyridine (DMAP) (0.25 g, 2.04 mmol) and trimethylamine (3 mL, 21.52 mmol). The solution was
stirred at reflux for 12 h, washed with hydrochloric acid (10% solution) (100 mL) and extracted with ethyl acetate (2 x 20 mL).
The crude was dried over sodium sulphate, rotoevaporated and purified by flash chromatography using a 1:1 mixture of
petroleum: acetone as eluent, to give 7 as a yellow solid. (1.96 g, 61%): mp = 90-91°C; 1H NMR  1.88 (m, 4H), 2.28 (t, J =
7.6 Hz, 4H), 2.37 (s, 6H), 2.43 (t, J = 6.8 Hz, 4H), 3.87 (s, 6H), 6.68 (d, J = 16.0 Hz, 2H), 7.03 (d, J = 2.0 Hz, Ar, 2H), 7.15 (d,
J = 2.0 Hz, Ar, 2H), 7.45 (d, J = 16.0 Hz, 2H); 13C NMR  19.72, 27.57, 32.46, 32.63, 56.10, 110.89, 123.03, 127.64, 131.38,
132.88, 139.27, 142.45, 151.70, 171.15, 178.37, 198.55.Anal. Calcd. for C32H43O12: C, 62.95; H, 5.61; Found: C, 62.73; H,
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5.64.
3,3'-dimethoxy-5,5'-bis((E)-3-oxobut-1-en-1-yl)-[1,1'-biphenyl]-2,2'-diyl dioctanoate (8)
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To a solution of 2 (0.9 g, 2.35 mmol) and triethylamine (0.51 g, 5.04 mmol) in dry dichloromethane (10 mL) octanoyl
chloride (0.96 g, 5.92 mmol) was added dropwise, at room temperature under nitrogen. The solution was stirred at room
temperature for 4 h, washed with water (100 mL) and extracted with dichloromethane (2 x 20 mL). The organic solution was
dried over sodium sulphate, rotoevaporated and purified by flash chromatography using a 2:1 mixture of petroleum : ethyl
1
acetate, as eluent, to give 8 as a white solid (1.31 g, 90%): mp =112-113°C; H NMR  0.87 (t, J = 6.8 Hz, 6H), 1.21-1.27
(series of m, 16.0 Hz), 1.52 (m, 4H), 2.35 (t, J = 7.6 Hz, 4H), 2.38 (s, 6H), 3.89 (s, 6H), 6.67 (d, J = 16.0 Hz, 2H), 7.05 (d, J =
2.0 Hz, Ar, 2H), 7.15 (d, J = 2 Hz, Ar, 2H), 7.47 (d, J = 16.0 Hz, 2H); 13C NMR  14.06, 22.59, 24.85, 27.59, 28.79, 28.92,
31.62, 33.79, 56.10, 110.72, 123.11, 127.51, 131.64, 132.63, 139.61, 142.33, 151.87, 171.07, 197.97.Anal. Calcd. for
C38H50O8: C, 71.90; H, 7.94; Found: C, 71.79; H, 7.84.
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O,O'-(3,3'-dimethoxy-5,5'-bis((E)-3-oxobut-1-en-1-yl)-[1,1'-biphenyl]-2,2'-diyl)diethyl disuccinate (9)
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To a solution of 2 (0.35 g, 0.92 mmol) and triethylamine (0.21 g, 2.07 mmol) in dry dichloromethane (8 mL), ethyl-3-
(chloroformyl)propionate (1.2 g, 7.29 mmol) was added, at room temperature under nitrogen. The solution was stirred at reflux
for 4 days, washed with water (100 mL) and extracted with dichloromethane (2 x 20 mL). The crude was dried over sodium
sulphate, rotoevaporated and purified by flash chromatography using a 1:1 mixture of petroleum : ethyl acetate, as eluent, to
give 9 as a white solid(0.45 g, 85%): mp = 97-98°C; 1H NMR  1.21 (t, J = 6.8 Hz, 6H), 2.37 (s, 6H), 2.55 (t, J = 6.8 Hz, 4H),
2.70 (t, J = 6.8 Hz, 4H), 3.88 (s, 6H), 4.10 (q, J = 6.8 Hz, 4H), 6.66 (d, J = 16.0 Hz, 2H), 7.01 (d, J = 2.0 Hz, Ar, 2H), 7.15 (d,
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J = 2.0 Hz, Ar, 2H), 7.48 (d, J = 16.0 Hz, 2H); 13C NMR  14.16, 27.52, 28.67, 28.96, 56.19, 60.66, 110.67, 122.26, 127:67,
131.19, 132.89, 139.29, 142.44, 151.79, 171.77, 198.15.Anal. Calcd. for C32H38O10: C, 65.97; H, 6.57; Found: C, 66.02; H,
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6.54.
(3E,3'E)-4,4'-(5,5'-dimethoxy-6,6'-bis(octadecyloxy)-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one) (10)
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To a solution of compound 2 (0.2 g, 0.52 mmol) and potassium carbonate (0.28 g, 2.13 mmol) and 18-crown-6 ether
(0.014 gr. 0.05 mmol) in dry acetonitrile (15 mL) 1-bromooctadecane (0.7 g, 2.15 mmol) was added dropwise at room
temperature under nitrogen. The solution was stirred at reflux for 12 h, acidified with hydrochloric acid (10% solution), extracted
with diethyl ether (2 x 50 mL) and dried over sodium sulphate. The crude product was then purified by silica chromatography
1
using a 2:1 mixture of petroleum : acetone, as eluent, to give 10 as a yellow solid (0.37 g. 80%): mp = 51-52°C; H NMR 
0.87 (t, J = 6.4 Hz, 6H), 1.07-1.27 (series of m, 64 H), 2.35 (s, 6H), 3.80 (t, J = 6.4 Hz, 4H), 3.91 (s. 6H), 6.64 (d, J = 16.4 Hz,
2H), 7.08 (d, J = 2.0 Hz, Ar, 2H), 7.11 (d, J = 2.0 Hz, Ar, 2H), 7.44 (d, J = 16.4 Hz, 2H). 13C NMR  14.06, 22.65, 25.74, 27.43,
29.30, 29.32, 29.60, 29.61, 29.63, 29.68, 30.06, 31.89, 55.92, 73.42, 110.72, 124.57, 126.23, 129.39, 132.67, 143.17, 148.62,
153.25, 198.06.Anal. Calcd. for C58H94O6: C, 78.50; H, 10.68; Found: C, 78.82; H, 10.72.
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(3E,3'E)-4,4'-(6,6'-bis(allyloxy)-5,5'-dimethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one) (11)
Starting from dehydrodivanillin, compound 20:
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To a solution of 20 (0.2 g, 0.52 mmol) in acetone (10 mL), aqueous 1N solution of sodium hydroxide (1.6 mL) was added and
the mixture was stirred at room temperature for 12 h. Water was added and, acidified with hydrochloric acid (10% solution) and
extracted with dichloromethane. The crude material was purified by flash chromatography using a 3:1 mixture of petroleum :
ethyl acetate, as eluent, to give 11 as a yellow solid (0,15 g, 65 %): mp 116-118 °C; 1H NMR  2.31 (s, 6H), 3.88 (s, 6H), 4.35
(m, 4H), 5.01 (m, 4H), 5.71 (m, 2H), 6.61 (d, J = 16.0 Hz, 2H), 7.05 (d, J = 2.0 Hz, Ar, 2H), 7.07 (d, J = 2.0 Hz, Ar, 2H), 7.42 (d,
J = 16.0 Hz, 2H);13C NMR  27.46, 55.92, 74.05, 110.74, 117.26, 124.31, 126.34, 129.68, 132.60, 133.96, 143.11, 147.98,
153.05, 198.21. Anal. Calcd for C28H30O6 C, 72,71; H, 6,54; Found: C, 72.21; H, 6.82.
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Starting from compound 2
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To a solution of compound 2 (0.15 g, 0.39 mmol) and potassium carbonate (0.12 g, 0.87 mmol) in dry acetone (10
mL), allyl bromide (0.1 g, 0.87 mmol) was added dropwise at room temperature under nitrogen. The solution was stirred at
reflux for 12 h, acidified with hydrochloric acid (10% solution), extracted with dichloromethane (2 x 50 mL) and dried over
sodium sulphate. The crude product was then purified by silica chromatography using a 2:1 mixture of petroleum : ethyl
acetate, as eluent, to give 11 (0.12g, 65%).
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(3E,3'E)-4,4'-(5,5'-dimethoxy-6,6'-bis((3-methylbut-2-en-1-yl)oxy)-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one) (12)
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To a solution of compound 2 (1 g, 2.64 mmol) and potassium carbonate (1.1 g, 7.8 mmol) in dry acetone (100 mL),
3,3ʹ-dimethylallyl bromide (1 g, 6.71 mmol) was added dropwise at room temperature under nitrogen. The solution was stirred
at reflux for 12 h, acidified with hydrochloric acid (10% solution), extracted with dichloromethane (2 x 50 mL) and dried over
sodium sulphate. The crude product was then purified by silica chromatography using a 2:1 mixture of petroleum : ethyl
acetate, as eluent, to give 12 as a yellow oil (0.8 g, 60%); 1H NMR  1.41 (s, 6H), 1.57 (s, 6H), 2.31 (s, 6H), 3.89 (s, 6H), 4.31
(d, J = 7.2 Hz, 2H), 5.17 (t, J = 1.6 Hz, 2H), 6.61 (d, J = 16.4 Hz, 2H), 7.06 (d, J = 2.0, Hz, Ar, 2H); 7.08 (d, J = 2.0, Hz, Ar,
2H); 7.43 (d, J = 16.4 Hz, 2H), 13C NMR  17.62, 25.72, 27.45, 55.94, 69.37, 110.57, 120.27, 124.60, 126.19, 129.49, 132.99,
138.20, 143.29, 148.14, 153.33, 198.22, Anal.Calcd for C32H38O6: C, 74.11; H, 7.39; Found: C, 74.69; H, 7.34.
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434
(3E,3'E)-4,4'-(6,6'-bis(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-5,5'-dimethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one)
(13)
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441
442
To a solution of 21 (0.4 g, 0.75 mmol) in acetone (8 mL) aqueous 1N solution of sodium hydroxide (0.5 mL) was
added and the mixture was stirred at room temperature for 12 h. Water was added and the solution was then acidified with
hydrochloric acid (10% solution) and extracted with dichloromethane. The crude material was purified by flash chromatography
using a 3:1 mixture of petroleum : ethyl acetate, as eluent, to give 13 as a yellow solid (0,04 g, 33%): mp 166-168 °C; 1H NMR
 1.45 (s, 6H), 1.54 (s, 6H), 1.66 (s, 6H), 1.86-1.98 (series of m, 8H), 2.35 (s, 6H), 3.93 (s, 6H), 4.38 (d, J = 6.8 Hz, 4H), 5.01
(m, 2H), 5.21 (m, 2H), 6.63 (d, J = 16.4 Hz, 2H), 7.09 (d, J = 2.0 Hz, Ar, 2H), 7.12 (d, J = 2.0 Hz, Ar, 2H), 7.46 (d, J = 16.4 Hz,
2H); 13C NMR  16.13, 17.63, 25.65, 26.26, 27.44, 39.52, 55.96, 69.53, 110.54, 119.89, 123.87, 124.61, 126.24, 129.52,
131.59, 133.02, 141.43, 143.28, 148.29, 153.35, 198.26. Anal. Calcd for C42H54O6 C, 77,03; H, 8,31; Found: C, 77.21; H, 8.52.
443
444
445
(E)-4-(4-(((2S,4R,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-3-methoxyphenyl)but-3-en-2-one
(18)
446
447
448
449
450
451
452
Compound 25 (1 g, 1.93 mmol) was stirred in sodium methoxide/methanol solution (0.02 g, 0.38 mmol in 20 ml) for
10 min. The reaction mixture was neutralized using a Dowex Marathon C (H+ form), filtrated and concentrated in vacuo to
obtain 18 as a yellow solid. (0.61 g, 95%): mp = 210-211°C; [α]D20 23.1 (c 0.5, MeOH); 1H NMR (CD3OD)  2.36 (s, 3H), 3.30-
3.65 (series of m, 4H), 3.71 (dd, J = 5.2, 17.2 Hz, 1H), 3.86 (m, 1H), 3.90 (s, 3H), 4.90 (s, 4H), 4.98 (d, J = 7.2 Hz, 1H), 6.70
(d, J = 16 Hz, 1H), 7.18 (d, J = 8.4 Hz, Ar, 1H), 7.21 (dd, J = 1.6, 8.4 Hz, Ar, 1H), 7.26 (d, J = 1.6 Hz, Ar, 1H), 7.59 (d, J = 16
Hz, 1H); 13C NMR  (CD3OD) 25.86, 55.36, 61.07, 69.87, 73.39, 76.45, 76.89, 100.76, 111.28, 116.01, 122.50, 125.14, 129.18,
144.19, 148.93, 149.65, 199.91.Anal. Calcd. for C17H22O7: C, 60.35; H, 6.55; Found: C, 60.39; H, 6.66
453
454
455
(E)-4-(3,4-dimethoxyphenyl)-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)but-3-en-2-
one (19)
456
457
458
459
460
461
462
Compound 26 (0.83 g, 1.54 mmol) was stirred in sodium methoxide/methanol solution (0.017 g, 0.32 mmol in 10 ml)
for 10 min. The reaction mixture was neutralized using a Dowex Marathon C (H+ form), filtrated and concentrated in vacuo to
obtain 19 as a brown solid. (0.54 g, 95%): mp = 152-154°C; [α]D20 -13.1 (c 0.25, MeOH); 1H NMR (CD3OD)  2.88 (dd, J = 9.2,
15.6 Hz, 1H), 3.12 (dd, J = 2.4, 15.6 Hz, 1H), 3.16 (d, J = 9.6, Hz, 1H), 3.24 (m, 1H), 3.33 (m, 2H), 3.60 (dd, J = 5.2, 12 Hz,
1H), 3.75 (m, 2H), 3.86 (s, 3H), 3.87 (s, 3H), 6.81 (d, J = 16.0 Hz, 1H), 6.98 (d, J = 8.4 Hz, Ar, 1H), 7.21 (dd, J = 2.0, 8.4 Hz,
Ar, 1H), 7.25 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 16.0 Hz, 1H); 13C NMR  (CD3OD) 42.73, 48.34, 54.91, 61.20, 70.11, 73.58,
76.01, 78.15, 80.05, 110.08, 111.05, 123.11, 123.81, 127.37, 143.85, 149.13, 151.50, 199.61.Anal. Calcd. for C18H24O8: C,
463
464
465
58.69; H, 6.57; Found: C, 58.80; H, 6.59.
6,6'-Bis-allyloxy-5,5'-dimethoxy-biphenyl-3,3'-dicarbaldehyde (20)
466
467
468
469
470
471
To a solution of dehydrodivanillin (0.68 g, 2.25 mmol) and potassium carbonate (0.8 g, 5.78 mmol) in dry acetone
(70 mL), allyl bromide (0.7 g, 5.78 mmol) was added dropwise, at room temperature under nitrogen. The solution was stirred
at reflux for 12 h, washed with water (100 mL) and extracted with ether (2 x 20 mL). The crude, was dried over sodium sulphate
to give 20 as a yellow solid (0.4 g, 50 %): mp = 86-88°C; 1H NMR  3.94 (s, 6H), 4.46 (d, J = 5.6 Hz, 4H), 4.96-5.00 (series of
m. 4H), 5.19-5.22 (series of m. 2H), 7.42 (d, J = 2.0 Hz, Ar, 2H), 7.47 (d, J = 2.0 Hz, Ar, 2H), 9.87 (s, 2H); 13C NMR  55.99,
69.61, 109.68, 123.76, 128.27, 131.81, 132.49, 141.84, 151.59, 153.69, 191.03. Anal. Calcd for C22H22O6: C, 69.10; H, 5.80;
472
Found: C, 69.15; H, 5.92.
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473
474
6,6'-bis(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-5,5'-dimethoxy-[1,1'-biphenyl]-3,3'-dicarbaldehyde (21)
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476
477
478
479
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481
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To a solution of dehydrodivanillin (0.52 g, 1.42 mmol) and potassium carbonate (0.59 g, 4.26 mmol) in dry acetone
(50 mL) dropwise geranyl bromide (1.0 g, 4.55 mmol) was added, at room temperature under nitrogen. The solution was
stirred at reflux for 12 h, washed with water (100 mL) and extracted with diethyl ether (2 x 20 mL). The crude, was dried over
sodium sulphate and then purified by silica chromatography using a 2:1 mixture of petroleum : ethyl acetate, as eluent, to give
21 as a yellow oil (0.71 g, 72%): 1H NMR  1.45 (s, 6H), 1.54 (s, 6H), 1.63 (s, 6H), 1.88-1.97 (series of m, 8H), 3.96 (s, 6H),
4.46 (d, J = 7.2 Hz, 4H), 4.96-5.00 (series of m, 2H), 5.19-5.22 (series of m, 2H); 7.42 (d, J = 2.0 Hz, Ar, 2H), 7.47 (d, J = 2.0
Hz, Ar, 2H), 9.87 (s, 2H); 13C NMR  16.14, 17.62, 25.62, 26.21, 39.47, 55.99, 69.61, 109.68, 119.63, 123.76, 128.27, 131.63,
131.81, 132.49, 141.84, 151.59, 153.69, 191.03. Anal. Calcd. for C36H46O6: C, 75,23; H, 8,07; Found: C, 75.69; H, 8.34.
483
484
485
(3E,3'E)-4,4'-(5,5'-dimethoxy-6,6'-bis(((2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-
[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one (22)
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487
488
489
490
491
492
493
494
Silver carbonate (3.52g, 12.8 mmol) was added to a stirred solution of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl
bromide (3 g, 7.29 mmol) and 2 (0.62 g, 1.62 mmol) in pyridine (30 mL) at room temperature under shaded conditions. After
stirring for 12 h, the solution was diluted with ethyl acetate (30 mL), washed with hydrochloric acid (10% solution) (100 mL),
dried over anhydrous sodium sulphate and rotoevaporated. The residue was purified by flash chromatography using a 1:2
mixture of petroleum : ethyl acetate as eluent, to give 22 as a yellow solid. (1.01 g, 60%): mp = 187-188°C; [α]D20 -11.5 (c 0.5,
CHCl3); 1H NMR  1.92 (s, 6H), 1.93 (s, 6H), 1.95 (s, 6H), 1.96 (s, 6H), 2.34 (s, 6H), 3.54 (m, 2H), 3.86, (m, 2H), 3.91 (s, 6H),
4.01 (m, 2H), 4.80-5.15 (series of m, 8H), 6.61 (d, J = 16 Hz, 2H), 6.97 (bs, Ar, 2H), 7.10 (d, J = 2 Hz, Ar, 2H), 7.39 (d, J = 16
Hz, 2H); 13C NMR  19.62, 19.64, 19.76, 19.81, 26.25, 55.88, 61.65, 68.32, 70.14, 71.45, 71.61, 72.46, 100.23, 111.35, 124.8,
126.92, 131.02, 142.36, 144.47, 152.47, 168.87, 169.06, 169.37, 169.76, 197.01; Anal.Calcd. for C50H58O24: C, 57.58; H, 5.61;
495
Found: C, 57.60; H, 5.66.
496
497
498
(S,R,S,R,R,3E,3'E)-4,4'-(5,5',6,6'-tetramethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(1-((2S,3R,4R,5S,6R)-3,4,5-triacetoxy-6-
(acetoxymethyl)tetrahydro-2H-pyran-2-yl)but-3-en-2-one) (24)
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500
501
502
503
504
505
506
507
508
509
510
To a solution of per-O-acetylated β-C-glucopyranosyl ketone (0.51 g, 1.32 mmol) and 2,2ʹ,3,3ʹ-tetramethoxy-5,5’-
diformyl-1,1ʹ-biphenyl 23 (OMe-dehydrodivanillin) (0.2 g, 0.62 mmol) in 2 mL dry dichloromethane, pyrrolidine (0.018 g, 0.24
mmol) was added under nitrogen. The reaction was stirred at room temperature until the starting material was consumed as
evidenced by TLC (48 h). The reaction mixture was neutralized with hydrochloric acid (10% solution) and the residue diluted
in dichloromethane. The organic extracts were combined, dried over sodium sulphate, filtered and evaporated. The crude
product was purified by column chromatography using a 2:3 mixture of petroleum : acetone as eluent, to give 24 as a yellow
solid. (0.49 g, 77%): mp = 130-131°C; [α]D20 -16.3 (c 0.1, CHCl3); 1H NMR  1.96 (s, 6H), 1.97 (s, 6H), 1.98 (s, 6H), 1.99 (s,
6H), 2.63 (dd, J = 3.2, 16.8 Hz, 2H), 2.98 (dd, J = 8.4, 16.8 Hz, 2H), 3.69 (s, 6H), 3.70 (m, 2H), 3.92 (s, 6H), 3.98 (dd, J = 2.4,
12.4 Hz, 2H), 4.09 (m, 2H), 4.23 (dd, J = 4.8, 12.4 Hz, 2H), 4.94 (t, J = 9.2 Hz, 2H), 5.04 (t, J = 9.2 Hz, 2H), 5.19 (t, J = 9.2
Hz, 2H), 6.62 (d, J = 16 Hz, 2H), 7.04 (d, J = 2.0 Hz, Ar, 2H), 7.19 (d, J = 2 Hz, Ar, 2H), 7.47 (d, J = 16 Hz, 2H); 13C NMR 
20.59, 20.61, 20.65, 20.71, 42.49, 55.92, 60.86, 61.98, 68.44, 71.64, 74.07, 74.13, 75.68, 111.02, 124.19, 125.52, 129.59,
132.29, 143.35, 149.23, 153.01, 169.56, 169.97, 170.23, 170.61, 195.95; Anal.Calcd. for C52H62O24: C, 58.31; H, 5.84; Found:
511
C, 58.37; H, 5.86.
512
513
514
(2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-methoxy-4-((E)-3-oxobut-1-en-1-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl
triacetate (25)
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516
517
518
519
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523
Silver carbonate (4.5 g, 16.31 mmol) was added to a stirred solution of tetra-O-acetylglucopyranosyl bromide (3.67
g, 8.91 mmol) and dehydrozingerone (0.78 g, 4.05 mmol) in pyridine (30 mL) at room temperature under shaded conditions.
After stirring for 18 h, the solution was diluted with ethyl acetate (30 mL), washed with hydrochloric acid (10% solution) dried
over anhydrous sodium sulphate and rotoevaporated. The residue was purified by flash chromatography using a 1:1 mixture
of petroleum : ethyl acetate as eluent, to give 25 as a brown solid. (1.88 g, 90%): mp = 189-190°C; [α]D20 -15.3 (c 0.5, CHCl3);
1H NMR  1.93 (s, 3H), 1.94 (s, 3H), 1.96 (s, 3H), 1.99 (s, 3H), 2.33 (s, 3H), 3.70 (m, 1H), 3.80 (s, 3H), 4.11 (dd, J = 2, 12 Hz,
1H), 4.24 (dd, J = 3.6, 12 Hz, 1H), 4.90-5.25 (series of m, 4H), 6.56 (d, J = 16 Hz, 1H), 6.98-7.08 (series of m, Ar, 3H), 7.40
(d, J = 2 Hz, Ar, 1H); 13C NMR  20.51, 20.54, 20.62, 20.95, 27.38, 56.01, 61.83, 68.25, 71.04, 72.02, 72.41, 100.12, 111.41,
119.32, 121.93, 126.49, 130.81, 142.79, 147.95, 150.71, 169.24, 169.35, 170.15, 170.46, 198.17; Anal.Calcd. for C25H30O12:
524
C, 57.47; H, 5.79; Found: C, 57.42; H, 5.76.
525
526
527
(2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-((E)-4-(3,4-dimethoxyphenyl)-2-oxobut-3-en-1-yl)tetrahydro-2H-pyran-3,4,5-triyl
triacetate (26)
528
529
530
531
532
533
534
535
536
537
538
539
To a solution of per-O-acetylated β-C-glucopyranosyl ketone (1.35 g, 3.47 mmol) and veratraldehyde (0.57 g, 3.47
mmol) in 10 mL dry dichloromethane, pyrrolidine (0.05 g, 0.64 mmol) was added under nitrogen. The reaction was stirred at
room temperature until the starting material was consumed as evidenced by TLC (72 h). The reaction mixture was neutralized
with hydrochloric acid (10% solution) and the residue diluted in dichloromethane. The organic extracts were combined, dried
over sodium sulphate, filtered and evaporated. The crude product was purified by column chromatography using a 1:1 mixture
of petroleum : acetone as eluent, to give 26 as a light yellow solid. (1.3 g, 70%): mp = 146-148°C; [α]D20 -25.3 (c 0.1, CHCl3);
1H NMR  1.91 (s, 3H), 1.92 (s, 3H), 1.94 (s, 3H), 1.95 (s, 3H), 2.58 (dd, J = 2.8, 16 Hz, 1H), 2.83 (dd, J = 8, 16 Hz, 1H), 3.68
(m, 1H), 3.83 (s, 3H), 3.85 (s, 3H), 3.93 (dd, J = 2, 12 Hz, 1H), 4.01 (m, 1H), 4.17 (dd, J = 5.2, 12.4 Hz, 1H), 4.89 (t, J = 9.6
Hz, 1H), 5.04 (t, J = 9.6 Hz, 1H), 5.19 (t, J = 9.6 Hz, 1H), 6.54 (d, J = 16 Hz, 1H), 6.80 (d, J = 8.4 Hz, Ar, 1H), 6.99 (d, J = 2
Hz, Ar, 1H), 7.05 (dd, J = 2, 8.4 Hz, 1H), 7.41 (d, J = 16 Hz, 1H); 13C NMR  20.09, 21.04, 42.81, 56.22, 56.36, 62.11, 68.85,
72.13, 74.92, 75.68, 75.89, 110.14, 111.6, 123.4, 124.33, 127.82, 143.81, 149.81, 152.13, 170.24, 171.85, 196.16; Anal.Calcd.
for C26H32O12: C, 58.20; H, 6.01; Found: C, 58.17; H, 6.06.
540
541
542
Biological procedures
Cell lines and Cell cultures
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Malignant melanoma cell lines used in this study [LCP-mel (LCP), LCM-mel (LCM), CN-mel (CN) and M14] were primary
tumor cell lines derived from tumor biopsy samples of malignant melanoma patients. They have been all kindly provided by the
Institute Dermopatico dell’Immacolata (IDI) in Rome. Cell lines had been all previously genetically characterized.[38] LCP was
derived from a primitive tumor, while LCM from a lymph node metastasis of the same melanoma patient. Both cell lines carry a
BRAF V600R mutation and a p16CD4N2A exon 2 deletion (LCP) or a p16 CD4N2A exons 1-2 deletion (LCM). CN cell line was derived
Q61R
from a melanoma lymph node metastasis and carries a NRAS
mutation. M14 cell line was derived from a melanoma
cutaneous metastasis, it was established in 1975 [29] and it is also commercially available (ATCC). It carries a BRAF V600E mutation
and an impaired locus CDKN2A (p16 455insC/del26 IVS1+2T>C).
A healthy donor human fibroblasts cell line (BJ) was used as normal cells control. It has been purchased from ATCC
(ATCC® CRL-2522).
Melanoma cells and fibroblasts were both grown in RPMI culture medium with stable glutamine, supplemented with 10%
Fetal Bovine Serum (FBS) and penicillin/streptomycin (1 U / mL) (complete medium) in a humidified atmosphere with 5% CO2, at
37 °C.
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Cell proliferation assay
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000709
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Cell proliferation assays were carried out for the 18 compounds, following the procedure previously described.[39] Briefly,
cells were plated in 96-well plates in complete medium at the density of 3.0 × 103 cells per well and incubated in a humidified
atmosphere with 5% CO2, at 37 °C. After 24h, medium was removed and replaced on days 1 and 3 by only fresh medium (control)
or by medium supplemented with increasing concentrations of the freshly prepared solution of compounds 2-19). After treatments
cell viability was determined on day 2 (24h) or day 4 (72h) by MTT test.[40] Briefly 20 L of MTT (5 mg/mL) were added to each
well. After an incubation of 3 h at 37 °C the medium was removed and formazan crystals were dissolved with 100 L DMSO per
well, for 10 min at room temperature with gentle mix. Absorbance was measured at 570 nm using a microplate reader (Sunrise™
Absorbance Reader - TECAN). Percentage of cell growth was calculated by normalizing the absorbance of treated cells to that of
the corresponding control. All the experiments were performed in triplicate and repeated at least three times.
568
Statistical analysis
569
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Relative IC50 values were determined by nonlinear regression of variable slope (four parameters) model by Graph Pad Prism
version 7.00 for Windows, Graph Pad Software, La Jolla California USA, www.graphpad.com. The average IC50 values, ± standard
deviation (SD) were calculated based on the results obtained from three independent experiments of proliferation assay. The
statistical significance of differential findings between experimental groups and controls was determined by Student's t-test. These
findings were considered significant if P values were < 0.001.
574
Supporting information
575
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The supporting information contains 1H NMR and 13C NMR spectra of compounds 3-13 and 18, 19.
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Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
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Keywords: cancer, drug discovery, Michael acceptor, molecular scaffold, natural products
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Cutaneous malignant melanoma is the most lethal form of skin cancer that arises from uncontrolled proliferation of melanocytes
that are cells producing pigments. We have prepared a small collection of hydroxylated biphenyls derivatives, compounds 2-13,
structurally related to a class of naturally occurring compounds known for selective and effective antiproliferative activity on
malignant melanoma cells.
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