Synthesis and antiangiogenic activity study of new hop chalcone Xanthohumol analogues

Article abstract of DOI:10.1016/j.ejmech.2017.07.024

Angiogenesis induction is a hallmark of cancer. Antiangiogenic properties of Xanthohumol (XN), a naturally occurring prenylated chalcone from hops, have been widely reported. Here we describe the synthesis and study the antiangiogenic activity in vitro of

Full text of DOI:10.1016/j.ejmech.2017.07.024

Accepted Manuscript
Synthesis and antiangiogenic activity study of new hop chalcone Xanthohumol
analogues
Elisa Nuti, Barbara Bassani, Caterina Camodeca, Lea Rosalia, AnnaRita Cantelmo,
Cristina Gallo, Denisa Baci, Antonino Bruno, Elisabetta Orlandini, Susanna Nencetti,
Douglas Noonan, Adriana Albini, Armando Rossello
PII:
S0223-5234(17)30546-9
10.1016/j.ejmech.2017.07.024
EJMECH 9587
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 March 2017
Revised Date: 14 July 2017
Accepted Date: 15 July 2017
Please cite this article as: E. Nuti, B. Bassani, C. Camodeca, L. Rosalia, A. Cantelmo, C. Gallo, D. Baci,
A. Bruno, E. Orlandini, S. Nencetti, D. Noonan, A. Albini, A. Rossello, Synthesis and antiangiogenic
activity study of new hop chalcone Xanthohumol analogues, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.07.024.
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Synthesis and Antiangiogenic Activity Study of New Hop Chalcone
Xanthohumol Analogues
Elisa Nuti,a,g,# Barbara Bassani,b,# Caterina Camodeca,a Lea Rosalia,a AnnaRita Cantelmo,c
Cristina Gallo,d Denisa Baci,b Antonino Bruno,b Elisabetta Orlandini,a,e,g Susanna Nencetti,a,g
Douglas Noonan,f Adriana Albini,b,§,* Armando Rossello a,g,§,*
a Dipartimento di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy.
b Laboratory of Vascular Biology and Angiogenesis, Scientific and Technologic Park, IRCCS
MultiMedica, via Fantoli 16/15, 20138 Milan, Italy.
c Laboratory of Angiogenesis and Vascular Metabolism, Vesalius Research Center, Center for
Cancer Biology (CCB), VIB, Leuven, Belgium.
d Laboratory of Translational Research, Arcispedale S. Maria Nuova-IRCCS, viale Risorgimento
80, 42121 Reggio Emilia, Italy.
e Dipartimento di Scienze della Terra, Università di Pisa, via Santa Maria 53, 56126 Pisa, Italy.
f Department of Biotechnologies and Life Sciencies, University of Insubria, Viale O. Rossi 9,
21100 Varese, Italy.
g Centro Interdipartimentale di Ricerca “Nutraceutica e Alimentazione per la Salute”, Università
di Pisa, Via del Borghetto 80, 56124 Pisa, Italy
* To whom correspondence should be addressed.
Adriana Albini: phone, +39 02 55406574; e-mail: adriana.albini@multimedica.it
Armando Rossello: phone, +39 050 2219562; fax, +39 050 2219605; e-mail:
armando.rossello@farm.unipi.it
# E.N. and B.B. contributed equally to this work.
§AA. and A.R. share equal authorship
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Abstract
Angiogenesis induction is a hallmark of cancer. Antiangiogenic properties of Xanthohumol
(XN), a naturally occurring prenylated chalcone from hops, have been widely reported. Here we
describe the synthesis and study the antiangiogenic activity in vitro of a series of XN derivatives,
where different substituents on the B-ring of the chalcone scaffold were inserted. The new XN
derivatives inhibited human umbilical-vein endothelial cell (HUVEC) proliferation, adhesion,
migration, invasion and their ability to form capillary-like structures in vitro at 10 µM
concentration. The preliminary results indicate that the phenolic OH group in R, present in
natural XN, is not necessary for having antiangiogenic activity. In fact, the most effective
compound from this series, 13, was characterized by a para-methoxy group in R and a fluorine
atom in R2 on B-ring. This study paves the way for future development of synthetic analogues of
XN to be used as cancer angiopreventive and chemopreventive agents.
Keywords:
xanthohumol analogues
prenylated chalcones
antiangiogenic activity
chemopreventive agents
Abbreviations: XN, xanthohumol; HUVEC, human umbilical vein endothelial cells; EGCG,
epigallocatechin-3-gallate;
MOMCl,
chloromethylmethyl
ether;
DEAD,
diethyl
azodicarboxylate; SAR, structure-activity relationship; FBS, foetal bovine serum; FITC,
fluorescein isothiocyanate; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride.
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1.
Introduction
Angiogenesis refers to the formation of new blood vessels from pre-existing vasculature [1].
Physiological angiogenesis is necessary for key processes such as wound healing and tissue
regeneration and repair. In pathological conditions, including cardiovascular diseases, diabetes,
cancer, and other pathologies associated with chronic inflammation, angiogenesis represent a
relevant hallmark contributing to disease insurgence and progression. Therefore, the induction of
new blood vessels is a necessary condition for tumors to recruit oxygen, nutrients and
disseminate to distant sites. Strategies aimed at blocking or delaying tumor angiogenesis, have
been employed both in therapeutic and (chemo) prevention settings and promising results have
been obtained in pre-clinical models and in the clinics [2, 3]. Cancer chemoprevention offers the
possibility of long-term low toxic treatments that could keep initial tumors at bay, delaying their
progression to a clinically relevant tumor [4-6]. Many chemopreventive agents are directly
derived from natural sources (phytochemicals) that have been reported to exert anti-proliferative,
pro-apoptotic, antioxidant and anti-inflammatory activities [7]. We have demonstrated that many
of these phytochemical derived agents act also as anti-angiogenic and anti-inflammatory drugs, a
concept we named “angioprevention” [8-10]. Substantial attention has been addressed to
flavonoids and their synthetic precursors, chalcones, a class of polyphenolic compounds that,
within their wide range of activity, also exhibit anti-angiogenic properties [11]. During the last
decades, the prenylated chalcone Xanthohumol (XN, 1, Fig. 1) has emerged as a cancer
chemopreventive agent [12]. It is the major prenylated chalcone present in the female
inflorescences of the hop plant Humulus lupulus L. (Cannabaceae) employed in the brewing
process to preserve and to add bitterness and flavor to beer. The beneficial properties of hops are
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well known from ancient times and have been used in traditional medicines since the IX century.
Xanthohumol was first isolated by Power in 1913 and its structure was elucidated in 1957 by
Verzele [13]. However, only in the last decades, we assisted to an increasing interest to this
molecule, since it is endowed with multiple biological activities including anti-diabetic, anti-
inflammatory, anti-oxidant, anti-cancer, anti-invasive, and anti-angiogenic activities [14]. Given
the promising results from in vitro and in vivo preclinical studies that have pointed out the
numerous benefits exerted by XN, clinical trials are currently being developed evaluating the
feasibility of XN treatment in the context of metabolic syndrome and prevention of DNA
damage (https://clinicaltrials.gov/) [15]. We showed that XN has stronger anti-angiogenic
activity as compared to the green tea flavonoid epigallocatechin-3-gallate (EGCG) [16]. Due to
the multiple beneficial activities of XN, we were interested in determining whether chemical
modification of the XN-scaffold resulted in more effective inhibition of angiogenesis, to identify
novel potential chemopreventive agents. Therefore, we developed of a series of novel synthetic
analogues of XN, compounds 2-14 [17], reported in Table 1. A series of XN analogues has been
recently synthesized and shown to have toxicity toward HeLa cells by inhibiting the
selenoprotein thioredoxin reductases (TrxRs) [18]. We chose to replace the phenolic group on B-
ring of the XN (Fig. 1) with several substituents endowed with different electronic and steric
properties such as halogens, methoxy or nitro groups, to evaluate the impact of these
modifications on antiangiogenic activity. The prenyl group on A-ring was left unchanged, since
previous studies [19] confirmed its importance for antiangiogenic activity. Derivatives 3, 5, 7, 10
and 13 were synthesized and tested together with some analogues MOM-protected on A-ring,
defined compounds 2, 4, 6, 8, 9, 11, 12 and 14. Here, we compared the antiangiogenic effects of
XN and our novel synthetic derivatives in vitro, where human umbilical vein endothelial cells
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(HUVEC) were used as a model for cell proliferation, apoptosis, cell adhesion, migration,
invasion and formation of capillary-like structures.
Fig. 1. Natural product Xanthohumol (XN) and its new synthetic analogues.
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Table 1. Structure of the new synthetic XN analogues 2-14.
Compd
R
Cl
R1 R2
R3
MOM
H
R4
H
H
H
H
H
H
Cl
Cl
H
H
H
H
F
H
H
H
H
F
2
3
Cl
F
MOM
H
4
F
5
H
MOM
H
6
H
F
7
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
NO2
H
H
H
F
MOM MOM
8
F
MOM
H
H
H
9
F
10
11
12
13
14
H
H
H
H
MOM MOM
F
MOM
H
H
H
H
F
H
MOM
2. Results and discussion.
2.1. Chemistry.
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The novel synthetic XN analogues have been synthesized following the synthetic approach
previously reported for the synthesis of XN and its natural derivatives [20, 21] and depicted in
Scheme 1.
OH
MOMO
OMOM
HO
OH
O
MOMO
OMOM
O
c
b
a
MOMO
OMOM
O
O
O
OH
OH
16
OH
15
17
18
d
MOMO
OH
R
R1
O
O
R2
MOMO
OMOM
O
MOMO
OMOM
R
2,4,6,9,12,14
e
f
R1
O
O
O
R2
8,11,20-23
20. R=Cl, R1=Cl, R2=H
19
HO
OH
R
R1
21. R=F,
22. R=H,
R1=H, R2=H
R1=H, R2=F
O
O
R2
23. R=NO2, R1=H, R2=H
3,5,7,10,13
Scheme 1. Synthesis of XN analogues 2-14. Reagents and conditions: (a) MOMCl, DIPEA, CH2Cl2,
rt, 6 h, 57%; (b) 3-methyl-2-butene-1-ol, DEAD, PPh3, THF, rt, 18 h, 60%; (c) N,N-dimethylaniline, 200
°C, 4 h, 42%; (d) (CH3O)SO2, K2CO3, acetone, reflux, 6 h, 65%; (e) the appropriate substituted
benzaldehyde, aqueous NaOH, MeOH, reflux, 4 h, 24-62%; (f) HCl, MeOH/H2O, 45 °C, 45 min, 7-77%.
Partial
methoxymethyl
(MOM)
protection
of
commercially
available
2,4,6-
trihydroxyacetophenone with chloromethylmethyl ether (MOMCl) yielded derivative 16. The
prenyl group has been then introduced using two subsequent steps: a Mitsunobu reaction with
prenyl alcohol to form the prenyl ether 17 followed by a Claisen rearrangement that provided
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prenylated acetophenone 18. The Mitsunobu condensation of phenol 16 with prenyl alcohol was
conducted in the presence of diethyl azodicarboxylate (DEAD) and triphenylphosphine (PPh3) in
THF (60% yield), then phenol 18 was obtained by treatment of 17 with N,N-dimethylaniline, at
200 °C for 4 h (42% yield). The phenolic hydroxyl group was then converted in the methoxy
function by reaction with dimethyl sulfate in the presence of K2CO3 (65%yield) and the
derivative 19 thus obtained was used in a Claisen-Schmidt condensation with diverse substituted
benzaldehydes to build up the chalcone scaffold. When 2,4 and 3,4-difluoro benzaldehydes have
been used, the chalcones obtained showed in 4 position a methoxy moiety, due to fluorine
displacement reaction occurring in the reaction conditions (NaOH aq. in MeOH, at 65 °C).
Finally, treatment of chalcones 20-23, 8, 11, with HCl in MeOH/H2O under controlled
temperature and acidity conditions, allowed to obtain the deprotected XN analogues 3, 5, 7, 10,
13 together with MOM mono-protected derivatives 2, 4, 6, 9, 12-14. Deprotected and mono-
protected derivatives were separated by flash chromatography.
2.2. Biological activity.
In preliminary experiments the anti-angiogenic activity of XN was compared to the one of green
tea epigallocatechin-3-gallate (EGCG), extensively described in literature as anti-angiogenic
flavonoid, by analyzing human endothelial cells proliferation, viability, cell functions, the ability
to interfere with migration, invasion and the capability to form capillary-like networks on matrix
basement membrane (data not shown) [16]. All our analysis pointed out a more powerful anti-
angiogenic-related activities of XN than EGCG. We therefore employed in all the assays
unmodified original Xantohumol molecule as a positive control for the synthetic derivatives.
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2.2.1. Effects of XN derivatives on HUVE cell proliferation and viability.
The newly synthesized compounds (4-14, Table 1) were first tested for cytotoxicity on human
umbilical vein endothelial cells (HUVECs). Compounds 2 and 3 were considered unfit for
testing due to some solubility issues. We treated HUVECs with increasing concentrations of XN
(the positive control for antiangiogenesis) and its derivatives ranging from 1 µM to 20 µM for
24, 48, 72 and 96 h. Cell viability was then assessed by the MTT assay and cell death was
demonstrated by Annexin-V/7-AAD double-staining and quantified on a flow cytometry. We
observed that most of the XN derivatives inhibited HUVEC proliferation in a dose-dependent
and time-dependent manner (Fig. 2). Since XN and many of its derivatives, such as 13 and 14
showed a relevant toxicity at 20 µM (Fig. 3), we used the 10 µM dose for the subsequent
experiments and consistent with previous publications [16, 22]. At the 10 µM concentration and
the 96h time point, several derivatives statistically inhibited HUVE cell growth as compared to
XN alone: in particular 8 and 11 (Fig. 2). Noteworthy, derivative 14 was the only compound to
show toxicity at 10 µM dose, as indicated by increased apoptosis on HUVECs (Fig. 3).
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Figure 2. Effects of XN derivatives on endothelial cell proliferation in vitro –XN and XN derivatives
interfere with endothelial cell (HUVEC) proliferation, as assessed by MTT assay. Results are showed as
Mean ± SEM; *p <0.05, ***p<0.001 compared to XN alone. “Vehicle” indicates cells treated with
vehicle (DMSO in the medium).
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Figure 3. Effects of XN derivatives on endothelial cell viability in vitro - Flow cytometry analysis
showed that 24 h treatment of endothelial cells with 20 µM XN derivatives resulted in increased rates of
apoptotic (AnnexinV+7-AAD-/+) cells. “Vehicle” indicates cells treated with vehicle (DMSO in the
medium). Results are showed as Mean ± SEM.
These results suggest that the presence of a MOM-protective group in R4 positively impacts on
the ability to block cell proliferation, considering that 8 and 11 (the only two derivatives of the
series bearing two MOM-protecting groups on A-ring) are the most potent inhibitors of HUVE
cell growth (Fig. 2). Moreover, the introduction of a strong electron-withdrawing group such as a
nitro group in R on B-ring seems to cause toxicity, as shown by the increased apoptosis on
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HUVECs given by compound 14 already at 10 µM concentration. Finally, only compound 12
characterized by a para-methoxy substituent on B-ring, a fluorine atom in R2 and a MOM-group
in R3 position was not able to affect endothelial cell growth.
2.2.2. Effect of XN derivatives on HUVE cell adhesion, migration and invasion
Since adhesion, migration and invasion are crucial steps for angiogenesis, we also investigated
the abilities of XN and its derivatives to inhibit these processes. We observed that XN and its
synthetic derivatives were able to decrease HUVEC adhesion compared with the nontreated
control (FBS 10% with dissolved vehicle, DMSO) (Fig. 4A). However, only one of the synthetic
derivatives, 13, was statistically more active than the parent compound XN. Comparable results
were obtained in terms of migration with XN and its derivatives (except for 12) which were able
to decrease HUVEC migration (Fig. 4B) compared with the control. Again, 13 showed
significantly greater inhibition than XN, along with compound 5. In the invasion assay, XN and
its derivatives (except for 9 and 12) showed statistical differences compared to that of the control
(Fig. 4C). However, in this case, none of the derivatives showed significantly more activity
compared to the parent compound XN.
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Figure 4. Effects of xanthohumol derivatives on HUVE cell adhesion, invasion and migration. XN
and XN derivatives at 10 µM concentration can interfere with crucial steps of angiogenesis by decreasing
HUVEC A) adhesion B) migration and C) invasion, as compared to vehicle-treated cells. “Vehicle”
indicates cells treated with vehicle (DMSO in the medium). Results are showed as Mean ± SEM. *p
<0.05; **p<0.001; ***p<0.001 as indicated by the bars.
These results point out the importance of a phenolic OH group in R3 together with a fluorine
atom on ring-B for having inhibitory activity on cell migration (see activity of 5 vs 4 and of 13
vs 12, Fig. 4B). In particular, compound 13, characterized by a para-methoxy substituent on B-
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ring and a fluorine atom in R2 position, showed the best activity on migration and adhesion
inhibition on HUVECs also compared to XN.
2.2.3. Effect of XN derivatives on morphogenesis in vitro
When cultured on a membrane-basement matrix layer, HUVE cells form capillary-like networks
within 6 h in vitro, mimicking the events occurring during vessel network formation in vivo. We
evaluated the ability of XN derivatives to effect HUVEC morphogenesis in vitro at 10 µM
concentration. We observed that all the XN derivatives were able to decrease the formation of
capillary-like structures in a statistically significant manner as compared to the control (FBS
10% without vehicle) (Fig. 5). Noteworthy, almost all XN derivatives were more potent than the
parental compound XN for all parameters considered, and among them 5, 8 and even more so 13
showed statistically significant differences in number of master segments.
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Figure 5. Effects of XN derivatives on HUVEC morphogenesis – Following 6 h treatment, XN and all
XN derivatives at 10 µM concentration can inhibit HUVEC ability to form capillary-like structures on
matrigel compared with vehicle-treated cells. SFM: cells treated with serum-free EGM-2 medium as a
negative control. FBS 10%: cells treated with medium containing 10% FBS without vehicle. “Vehicle”
indicates cells treated with vehicle (DMSO in the medium). Results are showed as Mean ± SEM. *p
<0.05; **p<0.001; ***p<0.001 as indicated by the bars.
Also in this assay, the best activity results were given by compound 13, characterized by a para-
methoxy substituent on B-ring and a fluorine atom in R2 position. Taken together, all these data
suggest that the new XN derivatives exert antiangiogenic activities that can be even higher than
the XN ones.
3. Conclusions.
A new series of synthetic derivatives of XN has been prepared and their effects on angiogenesis
have been evaluated in comparison with XN ones. In particular, compounds 4-14 have been
tested in vitro on cell proliferation, apoptosis, cell adhesion, migration, invasion and
morphogenesis with HUVE cells at 10 µM concentration. Despite the limited number of
derivatives synthesized in the present study, some comments about structure-activity
relationships (SAR) can be made. The results obtained so far suggest the importance of a
phenolic OH group in R3 on A-ring together with a fluorine atom on B-ring for having inhibitory
activity on cell migration (see activity of 5 vs 4 and of 13 vs 12, Fig. 4B). The presence of a
MOM-protective group in R4 seems to positively influence the ability to block cell proliferation,
considering that 8 and 11 are the most potent inhibitors of HUVE cell growth (Fig. 2). Moreover,
the introduction of a nitro group in R on B-ring seems to cause toxicity, as shown by the
increased apoptosis on HUVECs given by compound 14 already at 10 µM. Noteworthy, the
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phenolic OH group in R, present in natural XN, does not seem necessary for having
antiangiogenic activity. In fact, among all synthesized compounds, the best activity results were
exhibited by compound 13, which was the strongest inhibitor of cell adhesion, migration and
morphogenesis even compared to XN. This derivative is characterized by a para-methoxy group
in R and a fluorine atom in R2 on B-ring. Given the different activity between 13 and its
analogue bearing a fluorine in R1, compound 10, the position of F substitution on B-ring seems
to be determinant for activity.
Overall, these preliminary data highlight that the newly synthesized XN derivatives may
represent relevant compounds able to affect crucial steps of angiogenesis. Further studies must
be conducted to assess the pharmacokinetic properties of the most promising compounds in
comparison with naturally produced XN.
4. Experimental section
4.1. Chemistry. Melting points were determined on a Kofler hotstage apparatus and are
1
13
uncorrected. H, C and 19F NMR spectra were determined with a Varian Gemini 200 MHz
spectrometer or a Bruker Avance III HD 400 MHz spectrometer. Chemical shifts (δ) are reported
in parts per million and coupling constants (J) are given in hertz (Hz). The following
abbreviations are used: singlet (s), doublet (d), triplet (t), double-doublet (dd), double-triplet (dt),
broad (br) and multiplet (m). Chromatographic separations were performed on silica gel columns
by flash column chromatography (Kieselgel 40, 0.040–0.063 mm; Merck) or using ISOLUTE
Flash Si II cartridges (Biotage). Reactions were followed by thin-layer chromatography (TLC)
on Merck aluminum silica gel (60 F254) sheets that were visualized under a UV lamp.
Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was always used as
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the drying agent. Commercially available chemicals were purchased from Sigma-Aldrich (Milan,
Italy). Elemental analysis has been used to determine the purity of target compounds. Analytical
results are within ± 0.40% of the theoretical values.
4.2. 2′-Hydroxy-4′,6′-dimethoxymethylacetophenone (16).
To an ice-cooled suspension of 2′,4′,6′-trihydroxyacetophenone monohydrate (2.00 g, 10.74
mmol) in 21.50 mL of CH2Cl2, N,N-diisopropylethylamine (5.60 mL, 32.22 mmol) and then
dropwise MOMCl (2.40 mL, 32.22 mmol) were added. The reaction was gradually allowed to
reach room temperature and stirred for 6 h. The reaction was quenched with aqueous saturated
NH4Cl solution (20 mL). The resulting mixture was extracted with CH2Cl2/H2O (1:1). The
organic layers were combined, dried over Na2SO4, filtered, and evaporated. The yellowish
residue was purified by flash chromatography (n-hexane/EtOAc 6:1) using a Isolute Flash Si II
cartridge to afford 16 [23] as a colorless oil (1.57 g, 57% yield). 1H NMR (200 MHz, CDCl3) δ:
2.65 (s, 3H), 3.47 (s, 3H), 3.52 (s, 3H), 5.17 (s, 2H), 5.25 (s, 2H), 6.24 (d, J = 2.4 Hz, 1H), 6.27
(d, J = 2.4 Hz, 1H).
4.3. 4′,6′-Dimethoxymethyl-2′-(3-methylbut-2-en-1-yloxy)acetophenone (17).
To an ice-cooled solution of 16 (917 mg, 3.57 mmol) in 18 mL of dry THF, PPh3 (1.123 g,
4.30 mmol) and 3-methyl-2-buten-1-ol (0.54 mL, 5.35 mmol) were added. Diethyl
azodicarboxylate (DEAD) (0.90 mL, 5.71 mmol), was added dropwise and the resulting solution
was allowed to reach room temperature and stirred for 18 h. The solvent was evaporated, the
residue suspended in ether and the solid filtered. The filtrate was evaporated and the residue was
purified by silica gel flash chromatography (n-hexane/EtOAc 6:1) affording 17 [24] as a
colorless oil (684 mg, 60% yield). 1H NMR (200 MHz, CDCl3) δ: 1.70 (s, 3H), 1.75 (s, 3H), 2.47
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(s, 3H), 3.45 (s, 3H), 3.47 (s, 3H), 4.49 (d, J = 6.6 Hz, 2H), 5.12 (s, 2H), 5.15 (s, 2H), 5.40 (t, J =
6.6 Hz, 1H), 6.30 (d, J = 1.8 Hz, 1H), 6.43 (d, J = 1.8 Hz, 1H).
4.4. 2′,4′-Dimethoxymethyl-6′-hydroxy-3′-(3-methylbut-2-en-1-yloxy)-acetophenone (18).
Intermediate 17 (680 mg, 2 mmol) was dissolved in 21 mL of N,N-dimethylaniline and heated
at 200 °C for 4 h. Ethyl acetate (100 mL) was added and the mixture washed with 1 N HCl (2 ×
100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated. The residue was
purified by flash chromatography (n-hexane/EtOAc 20:1) using a Isolute Flash Si II cartridge to
1
afford 18 [24] as yellow oil (275 mg, 42% yield). H NMR (200 MHz, CDCl3) δ: 1.68 (s, 3H),
1.76 (s, 3H), 2.70 (s, 3H), 3.30 (d, J = 6.6 Hz, 2H), 3.45 (s, 3H), 3.51 (s, 3H), 4.95 (s, 2H), 5.12-
5.14 (m, 1H), 5.21 (s, 2H), 6.47 (s, 1H).
4.5. 2′,4′-Dimethoxymethyl-6′-methoxy-3′-(3-methylbut-2-en-1-yloxy)-acetophenone (19).
To a stirred solution of 18 (270 mg, 0.83 mmol) in 17 mL of acetone, K2CO3 (230 mg, 1.66
mmol) was slowly added and then dimethyl sulfate (0.15 mL, 1.66 mmol). The reaction mixture
was refluxed for 6 h, allowed to cool to room temperature, and quenched with 16 mL of NH4OH.
Then, it was extracted with CH2Cl2/H2O (1:1) 3 × 30 mL. The organic layers were combined,
dried over Na2SO4, filtered and evaporated. The residue was purified by flash chromatography
(n-hexane/EtOAc 8:1) using a Isolute Flash Si II cartridge to afford 19 [20] as yellow oil (183
mg, 65% yield). 1H NMR (200 MHz, CDCl3) δ: 1.66 (s, 3H), 1.74 (s, 3H), 2.49 (s, 3H), 3.30 (d,
J = 6.6 Hz, 2H), 3.47 (s, 3H), 3.48 (s, 3H), 3.79 (s, 3H), 4.90 (s, 2H), 5.12-5.14 (m, 1H), 5.20 (s,
2H), 6.54 (s, 1H).
4.6. General Procedure to Synthesize Chalcones 8, 11, 20-23.
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To a stirred solution of 19 (1 eq) and the properly substituted benzaldehyde (1 eq) in MeOH
(40 mL/mmol), 10% NaOH aqueous solution (2 mL/mmol) was added and the reaction mixture
was refluxed for 4 h. The solution was then extracted with EtOAc/H2O (1:1). The combined
organic layers were dried over Na2SO4, filtered, and evaporated. The residue was purified by
flash chromatography affording the desired compounds 8, 11, 20-23.
4.7.
(E)-3-(3-fluoro-4-methoxyphenyl)-1-(6-methoxy-2,4-bis(methoxymethoxy)-3-(3-
methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (8).
The title compound was prepared according the general procedure, using 3,4
difluorobenzaldehyde (42 mg, 0.29 mmol). The crude was purified by flash chromatography (n-
hexane/EtOAc 10:1) using a Isolute Flash Si II cartridge to afford 8 as yellow oil (57 mg, 41%
1
yield). H NMR (200 MHz, CDCl3) δ: 1.70 (s, 3H), 1.78 (s, 3H), 3.37 (d, J = 6.8 Hz, 2H), 3.45
(s, 3H), 3.52 (s, 3H), 3.79 (s, 3H), 3.94 (s, 3H), 4.93 (s, 2H), 5.15-5.22 (m, 1H), 5.26 (s, 2H),
13
6.61 (s, 1H), 6.89 (d, J = 15.7 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 7.25-7.38 (m, 3H). C NMR
(100 MHz, CDCl3) δ: 17.9, 22.1, 25.8, 55.9, 56.2, 56.2, 57.4, 89.3, 94.2, 101.0, 108.3, 109.0,
113.9, 114.3, 123.6, 126.4, 126.8, 129.0, 131.4, 141.0, 150.2, 153.6 (d, 1JC-F = 246.6 Hz)), 161.5,
162.0, 164.3, 193.0. Elemental Analysis for C26H31FO7 calculated: % C, 65.81; % H, 6.58;
found: % C, 65.93; % H, 6.76.
4.8.
(E)-3-(2-fluoro-4-methoxyphenyl)-1-(6-methoxy-2,4-bis(methoxymethoxy)-3-(3-
methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (11).
The title compound was prepared according the general procedure, using 2,4
difluorobenzaldehyde (114 mg, 0.80 mmol). The crude was purified by flash chromatography (n-
hexane/EtOAc 8:1) using a Isolute Flash Si II cartridge to afford 11 as yellow oil (118 mg, 31%
19

ACCEPTED MANUSCRIPT
1
yield). H NMR (200 MHz, CDCl3) δ: 1.68 (s, 3H), 1.77 (s, 3H), 3.36 (d, J = 6.6 Hz, 2H), 3.44
(s, 3H), 3.51 (s, 3H), 3.77 (s, 3H), 3.83 (s, 3H), 4.92 (s, 2H), 5.14-5.21 (m, 1H), 5.24 (s, 2H),
13
6.60 (s, 1H), 6.65-6.74 (m, 2H), 6.98 (d, J = 16.0 Hz, 1H), 7.46-7.56 (m, 2H). C NMR (100
MHz, CDCl3) δ: 18.0, 23.1, 25.8, 55.8, 56.1, 56.2, 57.6, 89.2, 94.5, 100.9, 101.9, 110.8, 111.1,
115.8, 122.8, 128.7, 130.0, 131.3, 136.7, 161.0, 162.6 (d, 1JC-F = 254.0 Hz), 162.6, 162.7, 164.3,
194.1. 19F NMR (376 MHz, CDCl3) δ: -111.32. Elemental Analysis for C26H31FO7 calculated: %
C, 65.81; % H, 6.58; found: % C, 66.17; % H, 6.63.
4.9. (E)-3-(3,4-dichlorophenyl)-1-(6-methoxy-2,4-bis(methoxymethoxy)-3-(3-methylbut-
2-en-1-yl)phenyl)prop-2-en-1-one (20).
The title compound was prepared according the general procedure, using 3,4-
dichlorobenzaldehyde (93 mg, 0.53 mmol). The crude was purified by flash chromatography (n-
hexane/EtOAc 12:1) using a Isolute Flash Si II cartridge to afford 20 as a semisolid yellow oil
(163 mg, 62% yield). 1H NMR (200 MHz, CDCl3) δ: 1.68 (s, 3H), 1.76 (s, 3H), 3.34 (d, J = 6.8
Hz, 2H), 3.41 (s, 3H), 3.50 (s, 3H), 3.77 (s, 3H), 4.90 (s, 2H), 5.14-5.21 (m, 1H), 5.24 (s, 2H),
6.59 (s, 1H), 6.96 (d, J = 15.9 Hz, 1H), 7.32 (d, J = 15.9 Hz, 1H), 7.38-7.47 (m, 2H), 7.60 (d, J =
1.8 Hz, 1H).
4.10. (E)-3-(4-fluorophenyl)-1-(6-methoxy-2,4-bis(methoxymethoxy)-3-(3-methylbut-2-
en-1-yl)phenyl)prop-2-en-1-one (21).
The title compound was prepared according the general procedure, using 4-
fluorobenzaldehyde (47 µL, 0.44 mmol). The crude was purified by flash chromatography (n-
hexane/EtOAc 12:1) using a Isolute Flash Si II cartridge to afford 21 as a semisolid yellow oil
1
(47 mg, 24% yield). H NMR (200 MHz, CDCl3) δ: 1.68 (s, 3H), 1.76 (s, 3H), 3.34 (d, J = 7.1
20

ACCEPTED MANUSCRIPT
Hz, 2H), 3.42 (s, 3H), 3.50 (s, 3H), 3.76 (s, 3H), 4.90 (s, 2H), 5.14-5.22 (m, 1H), 5.24 (s, 2H),
6.59 (s, 1H), 6.92 (d, J = 16.0 Hz, 1H), 7.06 (m, 2H), 7.38 (d, J = 16.0 Hz, 1H), 7.49-7.56 (m,
2H).
4.11. (E)-3-(2-fluorophenyl)-1-(6-methoxy-2,4-bis(methoxymethoxy)-3-(3-methylbut-2-
en-1-yl)phenyl)prop-2-en-1-one (22).
The title compound was prepared according the general procedure, using 2-fluorobenzaldehyde
(275 mg, 0.81 mmol). The crude was purified by flash chromatography (n-hexane/EtOAc 12:1)
1
using a Isolute Flash Si II cartridge to afford 22 as yellow oil (170 mg, 47% yield). H NMR
(200 MHz, CDCl3) δ: 1.68 (s, 3H), 1.79 (s, 3H), 3.35 (d, J = 6.9 Hz, 2H), 3.43 (s, 3H), 3.49 (s,
3H), 3.82 (s, 3H), 4.93 (s, 2H), 5.19-5.24 (m, 1H), 5.28 (s, 2H), 6.50 (s, 1H), 7.00-7.11 (m, 2H),
7.18 (d, J = 16.0 Hz, 1H), 7.29-7.36 (m, 1H), 7.50-7.60 (m, 2H).
4.12. (E)-1-(6-methoxy-2,4-bis(methoxymethoxy)-3-(3-methylbut-2-en-1-yl)phenyl)-3-(4-
nitrophenyl)prop-2-en-1-one (23).
The title compound was prepared according the general procedure, using 4-nitrobenzaldehyde
(201 mg, 1.33 mmol). The crude was purified by flash chromatography (n-hexane/EtOAc 6:1)
1
using a Isolute Flash Si II cartridge to afford 23 as yellow oil (274 mg, 44% yield). H NMR
(200 MHz, CDCl3) δ: 1.69 (s, 3H), 1.77 (s, 3H), 3.36 (d, J = 6.6 Hz, 2H), 3.42 (s, 3H), 3.48 (s,
3H), 3.79 (s, 3H), 4.92 (s, 2H), 5.16-5.24 (m, 1H), 5.26 (s, 2H), 6.61 (s, 1H), 7.11 (d, J = 16.2
Hz, 1H), 7.48 (d, J = 16.2 Hz, 1H), 7.66-7.71 (m, 2H), 8.20-8.26 (m, 2H).
4.13. General Procedure to Synthesize Xanthohumol analogues 2-7, 9, 10, 12-14.
21

ACCEPTED MANUSCRIPT
To a stirred solution of chalcone in MeOH/H2O, concentrated HCl was added until pH 1 and
the mixture was heated at 45 °C for 45 min. The reaction mixture was then extracted with
EtOAc/H2O, the organic layers were combined, dried over Na2SO4, filtered, and evaporated at 20
°C. The residue was purified by flash chromatography affording the desired compounds 2-7, 9,
10, 12-14.
4.14.
(E)-3-(3,4-dichlorophenyl)-1-(2-hydroxy-6-methoxy-4-(methoxymethoxy)-3-(3-
methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (2) and (E)-3-(3,4-dichlorophenyl)-1-(2,4-
dihydroxy-6-methoxy-3-(3-methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (3).
The title compounds were prepared according the general procedure, starting from compound
20 (62 mg, 0.12 mmol). The residue was purified by flash chromatography (n-hexane/EtOAc
7:1) using a Isolute Flash Si II cartridge to afford 2 (31 mg, 57% yield) as orange solid and 3 (9
1
mg, 18% yield) as yellow solid. (2) mp 105-107 °C. H NMR (400 MHz, CDCl3) δ: 1.67 (s, 3H),
1.79 (s, 3H), 3.32 (d, J = 7.1 Hz, 2H), 3.49 (s, 3H), 3.92 (s, 3H), 5.19-5.24 (m, 1H), 5.27 (s, 2H),
6.24 (s, 1H), 7.40 (dd, J1 = 8.3 Hz, J2 = 2.0 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 15.6
13
Hz, 1H), 7.65 (d, J = 2.0 Hz, 1H), 7.81 (d, J = 15.6 Hz, 1H). C NMR (100 MHz, CDCl3) δ:
17.7, 21.6, 25.8, 55.9, 56.2, 89.1, 93.9, 106.9, 111.0, 122.5, 127.3, 129.6, 129.6, 130.8, 131.4,
133.1, 133.6, 135.7, 138.9, 160.9, 161.3, 164.3, 192.5. Elemental Analysis for C23H24Cl2O5
1
calculated: % C, 61.21; % H, 5.36; found: % C, 61.49; % H, 5.48. (3) mp 62-64 °C. H NMR
(400 MHz, CDCl3) δ: 1.78 (s, 3H), 1.83 (s, 3H), 3.40 (d, J = 6.3 Hz, 2H), 3.90 (s, 3H), 5.26-5.32
(m, 1H), 5.95 (s, 1H), 6.26 (br, 1H), 7.39-7.48 (m, 2H), 7.60-7.66 (m, 2H), 7.84 (d, J = 15.8 Hz,
13
1H). C NMR (100 MHz, CDCl3) δ: 18.0, 21.7, 25.9, 56.0, 91.4, 106.2, 106.4, 121.7, 127.4,
129.7, 129.8, 130.9, 133.2, 133.8, 135.9, 136.3, 139.1, 161.3, 162.5, 165.3, 192.3. Elemental
Analysis for C21H20Cl2O4 calculated: % C, 61.93; % H, 4.95; found: % C, 62.24; % H, 5.12.
22

ACCEPTED MANUSCRIPT
4.15.
(E)-3-(4-fluorophenyl)-1-(2-hydroxy-6-methoxy-4-(methoxymethoxy)-3-(3-
methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (4) and (E)-1-(2,4-dihydroxy-6-methoxy-3-(3-
methylbut-2-en-1-yl)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (5).
The title compounds were prepared according the general procedure, starting from compound 21
(91 mg, 0.20 mmol). The residue was purified by flash chromatography (n-hexane/EtOAc 7:1)
using a Isolute Flash Si II cartridge to afford 4 (6 mg, 7% yield) as orange solid and 5 (29 mg,
41% yield) as yellow solid. (4) mp 80-82 °C. 1H NMR (400 MHz, CDCl3) δ: 1.67 (s, 3H), 1.79
(s, 3H), 3.33 (d, J = 7.1 Hz, 2H), 3.50 (s, 3H), 3.92 (s, 3H), 5.20-5.24 (m, 1H), 5.27 (s, 2H), 6.24
(s, 1H), 7.07-7.11 (m, 2H), 7.56-7.60 (m, 2H), 7.72 (d, J = 15.6 Hz, 1H), 7.79 (d, J = 15.6 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ: 17.9, 21.8, 25.9, 56.0, 56.3, 89.3, 94.0, 107.2, 111.1, 116.1
2
1
(d, JC-F = 21.8 Hz), 122.7, 127.7, 130.2, 131.4, 132.0, 140.8, 161.0, 161.2, 163.8 (d, JC-F
=
250.9 Hz), 164.4, 193.1. Elemental Analysis for C23H25FO5 calculated: % C, 68.99; % H, 6.29;
found: % C, 69.13; % H, 6.13. (5) mp 167-169 °C. 1H NMR (400 MHz, CDCl3) δ: 1.77 (s, 3H),
1.83 (s, 3H), 3.40 (d, J = 7.2 Hz, 2H), 3.89 (s, 3H), 5.27-5.31 (m, 1H), 5.95 (s, 1H), 6.30 (br,
1H), 7.07-7.11 (m, 2H), 7.56-7.60 (m, 2H), 7.73 (d, J = 15.6 Hz, 1H), 7.82 (d, J = 15.6 Hz, 1H).
2
13C NMR (100 MHz, CDCl3) δ: 18.0, 21.7, 25.9, 55.9, 91.3, 106.2, 106.5, 116.1 (d, JC-F = 21.9
1
Hz), 121.8, 127.7, 130.2, 132.0, 136.0, 140.9, 161.3, 162.2, 163.9 (d, JC-F = 250.0 Hz), 165.3,
192.8. Elemental Analysis for C21H21FO4 calculated: % C, 70.77; % H, 5.94; found: % C, 70.99;
% H, 5.70.
4.16.
(E)-3-(2-fluorophenyl)-1-(2-hydroxy-6-methoxy-4-(methoxymethoxy)-3-(3-
methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (6) and (E)-1-(2,4-dihydroxy-6-methoxy-3-(3-
methylbut-2-en-1-yl)phenyl)-3-(2-fluorophenyl)prop-2-en-1-one (7).
23

ACCEPTED MANUSCRIPT
The title compounds were prepared according the general procedure, starting from compound 22
(170 mg, 0.39 mmol). The residue was purified by flash chromatography (n-hexane/EtOAc 10:1)
using a Isolute Flash Si II cartridge to afford 6 (86 mg, 54% yield) and 7 (13 mg, 9% yield) as
yellow-orange solids. (6) mp 91-93 °C. 1H NMR (400 MHz, CDCl3) δ: 1.67 (s, 3H), 1.79 (s, 3H),
3.33 (d, J = 7.1 Hz, 2H), 3.49 (s, 3H), 3.91 (s, 3H), 5.20-5.25 (m, 1H), 5.27 (s, 2H), 6.24 (s, 1H),
7.08-7.19 (m, 2H), 7.31-7.37 (m, 1H), 7.55-7.60 (m, 1H), 7.83 (d, J = 15.8 Hz, 1H), 7.97 (d, J =
13
15.8 Hz, 1H). C NMR (100 MHz, CDCl3) δ: 17.9, 21.7, 25.9, 55.9, 56.3, 89.2, 94.0, 107.2,
2
111.1, 116.3 (d, JC-F = 22.0 Hz), 122.8, 123.8, 124.5, 129.8, 130.7, 131.3, 131.4, 134.6, 161.1,
1
161.3, 161.8 (d, JC-F = 254.0 Hz), 164.4, 193.3. 19F NMR (376 MHz, CDCl3) δ: -113.85.
Elemental Analysis for C23H25FO5 calculated: % C, 68.99; % H, 6.29; found: % C, 69.28; % H,
6.62. (7) mp 157-159 °C. 1H NMR (400 MHz, CDCl3) δ: 1.77 (s, 3H), 1.83 (s, 3H), 3.39 (d, J =
7.2 Hz, 2H), 3.88 (s, 3H), 5.26-5.30 (m, 1H), 5.94 (s, 1H), 6.40 (br, 1H), 7.08-7.19 (m, 2H),
7.31-7.37 (m, 1H), 7.56 -7.60 (m, 1H), 7.84 (d, J = 15.8 Hz, 1H), 8.00 (d, J = 15.8 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δ: 18.0, 21.7, 25.9, 55.8, 91.3, 106.3, 106.5, 116.3 (d, 2JC-F = 22.0 Hz),
1
121.8, 123.8, 124.5, 129.8, 130.6, 131.3, 134.7, 135.8, 161.4, 161.7 (d, JC-F = 253.9 Hz), 162.3,
165.3, 192.9. 19F NMR (376 MHz, CDCl3) δ: -113.89. Elemental Analysis for C21H21FO4
calculated: % C, 70.77; % H, 5.94; found: % C, 70.54; % H, 5.73.
4.17. (E)-3-(3-fluoro-4-methoxyphenyl)-1-(2-hydroxy-6-methoxy-4-(methoxymethoxy)-3-
(3-methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (9) (E)-1-(2,4-dihydroxy-6-methoxy-3-(3-
methylbut-2-en-1-yl)phenyl)-3-(3-fluoro-4-methoxyphenyl)prop-2-en-1-one (10).
The title compounds were prepared according the general procedure, starting from compound
8 (275 mg, 0.58 mmol). The residue was purified by silica gel flash chromatography (n-
hexane/EtOAc 4:1) to afford 9 (24 mg, 10% yield) and 10 (111 mg, 50% yield) as yellow-orange
24

ACCEPTED MANUSCRIPT
solids. (9) mp 89-91 °C. 1H NMR (400 MHz, CDCl3) δ: 1.67 (s, 3H), 1.79 (s, 3H), 3.32 (d, J =
7.1 Hz, 2H), 3.49 (s, 3H), 3.93 (s, 6H), 5.21-5.23 (m, 1H), 5.27 (s, 2H), 6.24 (s, 1H), 6.97 (dd,
JH-F = 8,5 Hz, JH-H = 8,5 Hz, 1H), 7.28-7.31 (m, 1H), 7.36 (dd JH-F = 12.2 Hz, JH-H = 2.0 Hz, 1H),
7.68 (d, J = 15.6 Hz, 1H), 7.74 (d, J = 15.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 17.9, 21.8,
2
25.9, 56.0, 56.4, 56.4, 89.3, 94.1, 107.2, 111.1, 113.3, 114.6 (d, JC-F = 18.5 Hz), 122.8, 126.1,
1
19
126.9, 129.1, 131.4, 140.9, 149.4, 152.6 (d, JC-F = 246.6 Hz), 161.0, 161.2, 164.4, 193.0. F
NMR (376 MHz, CDCl3) δ: -134.56. Elemental Analysis for C24H27FO6 calculated: % C, 66.96;
% H, 6.32; found: % C, 66.71; % H, 6.04. (10) mp 163-165 °C. 1H NMR (400 MHz, Acetone-d6)
δ: 1.63 (s, 3H), 1.76 (s, 3H), 3.28 (d, J = 7.2 Hz, 2H), 3.93 (s, 3H), 3.95 (s, 3H), 5.23-5.26 (m,
1H), 6.15 (s, 1H), 7.20 (dd JH-F = 8.6 Hz, JH-H = 8.5 Hz, 1H), 7.48-7.51 (m, 1H), 7.54 (dd JH-F
=
12.1 Hz, JH-H = 2.1 Hz, 1H), 7.69 (d, J = 15.5 Hz, 1H), 7.90 (d, J = 15.5 Hz, 1H), 9.23 (s, 1H).
13C NMR (100 MHz, Acetone-d6) δ: 17.8, 22.0, 25.8, 56.2, 56.6, 91.6, 106.3, 108.8, 114.5,
19
115.3, 115.5, 123.9, 126.9, 127.6, 129.7, 131.0, 141.4, 154.4, 161.9, 163.0, 166.3, 193.0. F
NMR (376 MHz, CDCl3) δ: -135.91. Elemental Analysis for C22H23FO5 calculated: % C, 68.38;
% H, 6.00; found: % C, 68.44; % H, 5.85.
4.18. (E)-3-(2-fluoro-4-methoxyphenyl)-1-(2-hydroxy-6-methoxy-4-(methoxymethoxy)-3-
(3-methylbut-2-en-1-yl)phenyl)prop-2-en-1-one (12) and (E)-1-(2,4-dihydroxy-6-methoxy-3-
(3-methylbut-2-en-1-yl)phenyl)-3-(2-fluoro-4-methoxyphenyl)prop-2-en-1-one (13).
The title compounds were prepared according the general procedure, starting from compound 11
(100 mg, 0.21 mmol). The residue was purified by silica gel flash chromatography (n-
hexane/EtOAc 7:1) to afford 12 (41 mg, 45% yield) as orange solid and 13 (15 mg, 18% yield)
as yellow-orange amorphous solid. (12) mp 94-96 °C. 1H NMR (400 MHz, CDCl3) δ: 1.66 (s,
3H), 1.78 (s, 3H), 3.31 (d, J = 7.1 Hz, 2H), 3.48 (s, 3H), 3.83 (s, 3H), 3.89 (s, 3H), 5.19-5.23 (m,
25

ACCEPTED MANUSCRIPT
1H), 5.25 (s, 2H), 6.22 (s, 1H), 6.64 (dd, JH-F = 12.5 Hz, JH-H = 2.5 Hz, 1H), 6.72 (dd, J1 = 8.6 Hz,
J2 = 2.5 Hz, 1H), 7.49 (dd, JH-F = 8.7 Hz, JH-H = 8.6 Hz, 1H), 7.80 (d, J = 15.8 Hz, 1H), 7.87 (d, J
13
= 15.8 Hz, 1H). C NMR (100 MHz, CDCl3) δ: 17.9, 21.8, 25.9, 55.8, 55.9, 56.3, 89.3, 94.1,
2
102.1 (d, JC-F = 26.0 Hz), 107.3, 110.8, 111.1, 116.6, 122.9, 128.1, 130.8, 131.4, 135.2, 161.0,
19
162.4, 162.5, 162.9 (d, 1JC-F = 254.4 Hz), 164.4, 193.3. F NMR (376 MHz, CDCl3) δ: -110.93.
Elemental Analysis for C24H27FO6 calculated: % C, 66.96; % H, 6.32; found: % C, 67.15; % H,
1
6.06. (13) H NMR (400 MHz, Acetone-d6) δ: 1.65 (s, 3H), 1.77 (s, 3H), 3.30 (d, J = 7.1 Hz,
2H), 3.91 (s, 3H), 3.93 (s, 3H), 5.23-5.26 (m, 1H), 6.83 (dd, JH-F = 12.8 Hz, JH-H = 2.5 Hz, 1H),
6.88 (dd, J1 = 8.4 Hz, J2 = 2.5 Hz, 1H), 7.74-7.78 (m, 1H), 7.85 (d, J = 15.7 Hz, 1H), 8.02 (d, J =
13
15.7 Hz, 1H) 9.03 (br, 1H). C NMR (100 MHz, Acetone-d6) δ: 16.9, 21.1, 24.9, 55.2, 55.4,
2
90.8, 101.6 (d, JC-F = 26.0 Hz), 105.4, 108.0, 111.2, 115.8, 122.9, 127.5, 130.1, 130.2, 133.8,
19
161.0, 162.5 (d, 1JC-F = 252.0 Hz), 162.7, 162.8, 165.5, 192.2. F NMR (376 MHz, CDCl3) δ: -
114.07. Elemental Analysis for C22H23FO5 calculated: % C, 68.38; % H, 6.00; found: % C,
68.49; % H, 6.31.
4.19.
(E)-1-(2-hydroxy-6-methoxy-4-(methoxymethoxy)-3-(3-methylbut-2-en-1-
yl)phenyl)-3-(4-nitrophenyl)prop-2-en-1-one (14)
Starting from compound 23 (274 mg, 0.58 mmol) and following the general procedure, MOM
mono-protected derivative 14 was obtained as the only reaction product. The residue was
purified by flash chromatography (n-hexane/EtOAc 7:1) using a Isolute Flash Si II cartridge to
1
afford 14 (191mg, 77% yield) as yellow-orange solids, mp 125-127 °C. H NMR (400 MHz,
CDCl3) δ: 1.67 (s, 3H), 1.79 (s, 3H), 3.33 (d, J = 7.1 Hz, 2H), 3.50 (s, 3H), 3.94 (s, 3H), 5.19-
5.25 (m, 1H), 5.28 (s, 2H), 6.26 (s, 1H), 7.70-7.74 (m, 3H), 7.94 (d, J = 15.7 Hz, 1H), 8.24-8.27
13
(m, 2H). C NMR (100 MHz, CDCl3) δ: 17.9, 21.7, 25.9, 56.1, 56.4, 89.3, 94.1, 107.1, 111.3,
26

ACCEPTED MANUSCRIPT
122.6, 124.3, 128.8, 131.6, 132.1, 138.5, 142.1, 148.3, 161.1, 161.7, 164.5, 192.5. Elemental
Analysis for C23H25NO7 calculated: % C, 64.63; % H, 5.90; % N, 3.28; found: % C, 64.47; % H,
5.77; % N, 3.44.
4.20. Cell culture
Human umbilical vein endothelial cells (HUVECs) were purchased by PromoCell (Heidelberg,
Germany) and cultured in endothelial cell growth medium-2 (EGM-2) (Lonza, Basel,
Switzerland) containing 10% FBS, 1% L-glutamine 2 mM and 1% Penicillin/Streptomycin
(Sigma-Aldrich, Milan, Italy). Cells were treated with XN (Sigma-Aldrich, Milan, Italy) and its
derivatives dissolved in DMSO in a stock solution of 20 mM and used at indicated
concentrations for different time periods. In all biological assays XN was used as positive
control.
4.21. Cell Proliferation assay
The effect of XN and its derivatives on HUVE cell proliferation was determined by 3-(4,5-
dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma Aldrich) assay. Briefly, 1 x
103 cells were seeded into 96-well plates and incubated at 37 °C overnight. Cells were then
treated with XN and its derivative with different concentrations ranging from 1 µM to 20 µM
and incubated for 24, 48, 72 or 96 h. Control cells (CTRL) received EGM-2 medium containing
10% FBS, 1% L-glutamine 2 mM and 1% Penicillin/Streptomycin with dissolved vehicle
(DMSO). MTT reagent 5 (mg/mL) was added to cell cultures and incubated for 3 hours at 37 °C.
After incubation, medium was replaced with 100 ꢀL of DMSO and the amount of solubilized
formazan was quantified at 540 nm in a SpectraMax M2 (Molecular Devices, Sunnyvale CA).
4.22. Determination of apoptosis by Flow Cytometry
27

ACCEPTED MANUSCRIPT
HUVECs (2 x 105 cells/well) were treated with increasing concentrations of XN and its synthetic
derivatives (ranging from 5 µM to 20 µM) for 24h. Control cells received EGM-2 medium
containing 10% FBS, 1% L-glutamine 2 mM and 1% Penicillin/Streptomycin with dissolved
vehicle (DMSO). Cells were then collected and stained with FITC-conjugated Annexin V (BD:
Becton Dickinson Bioscences, San Jose, CA) and 7-AAD (BD). Untreated cells and those treated
with DMSO (vehicle) were used as control. This method allows discrimination between viable
cells (Annexin V negative, 7-AAD negative) and apoptotic cells (Anexin V positive, 7-AAD
positive/negative). Analysis was performed on 20000 gated cells, excluding cell debris, using a
FACSCanto II (BD), with excitation set at 488 nm and emission at 518 nm (FITC detector) or
620 nm (PE fluorescence detector).
4.23. Cell adhesion assay in vitro
HUVECs (3x103 cells) were treated with XN and its derivatives (10 µM) and seeded in a 48
wells plate pre-coated for 45 min with fibronectin (2 µg/mL). Control cells received EGM-2
medium containing 10% FBS, 1% L-glutamine 2 mM and 1% Penicillin/Streptomycin with
dissolved vehicle (DMSO). After 90 min incubation, the supernatants were removed and cells
were washed with PBS. Cells were then fixed with 4% paraformaldehyde and stained with
DAPI. Six different replicates for each condition were considered. Eighteen microscope fields
were randomly selected from the six wells for each treatment to count the number of adherent
cells.
4.24. HUVEC migration and invasion in vitro assays
Chemoinvasion and chemotaxis assays were performed in modified Boyden chambers, as
previously described [25, 26]. Briefly, viable HUVE cells (5 x 104) were seeded in the upper
compartment of the Boyden chamber and treated with XN and its derivatives (10 µM) for 6 h
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