Synthesis and antiproliferative activity of minor hops prenylflavonoids and new insights on prenyl group cyclization

Article abstract of DOI:10.3390/molecules23040776

Synthesis of minor prenylflavonoids found in hops and their non-natural derivatives were performed. The antiproliferative activity of the obtained compounds against some human cancer cell lines was investigated. Using xanthohumol isolated from spent hops

Full text of DOI:10.3390/molecules23040776

molecules
Article
Synthesis and Antiproliferative Activity of Minor
Hops Prenylflavonoids and New Insights on Prenyl
Group Cyclization
Jarosław Popłon´ski 1,*, Eliza Turlej 2, Sandra Sordon 1, Tomasz Tronina 1
,
ID
Agnieszka Bartman´ska 1, Joanna Wietrzyk 2 and Ewa Huszcza 1
ID
1
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25,
50-375 Wrocław, Poland; Sandra.sordon@upwr.edu.pl (S.S.); Tomasz.tronina@upwr.edu.pl (T.T.);
Agnieszka.bartmanska@upwr.edu.pl (A.B.); Ewa.Huszcza@upwr.edu.pl (E.H.)
Department of Experimental Oncology, Hirszfeld Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland; elizaturlej@interia.pl (E.T.);
wietrzyk@iitd.pan.wroc.pl (J.W.)
2
*
Correspondence: jaroslaw.poplonski@upwr.edu.pl; Tel.: +48-71-320-5197
Academic Editor: Angela Rampa
Received: 7 March 2018; Accepted: 27 March 2018; Published: 28 March 2018
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Synthesis of minor prenylflavonoids found in hops and their non-natural derivatives
were performed. The antiproliferative activity of the obtained compounds against some human
cancer cell lines was investigated. Using xanthohumol isolated from spent hops as a lead compound,
a series of minor hop prenylflavonoids and synthetic derivatives were obtained by isomerization,
cyclisation, oxidative-cyclisation, oxidation, reduction and demethylation reactions. Three human
cancer cell lines—breast (MCF-7), prostate (PC-3) and colon (HT-29)—were used in antiproliferative
assays, with cisplatin as a control compound. Five minor hop prenyl flavonoids and nine non-natural
derivatives of xanthohumol have been synthetized. Syntheses of xanthohumol K, its dihydro- and
tetrahydro-derivatives and 100,200,
All of the minor hops prenyl flavonoids exhibited strong to moderate antiproliferative activity in vitro
α,β-tetrahydroxanthohumol C were described for the first time.
.
The minor hops flavonoids xanthohumol C and 100,200-dihydroxanthohumol K and non-natural
2,3-dehydroisoxanthohumol exhibited the activity comparable to cisplatin. Results described in the
article suggest that flavonoids containing chromane- and chromene-like moieties, especially chalcones,
are potent antiproliferative agents. The developed new efficient, regioselective cyclisation reaction of
the xanthohumol prenyl group to 100,200-dihydroxantohumol K may be used in the synthesis of other
compounds with the chromane moiety.
Keywords: xanthohumol; isoxanthohumol; chalcone; cyclisation; antiproliferative activity; hops;
spent hops
1. Introduction
Flavonoids are a group of secondary plant metabolites and important food constituents with a
daily intake of a few hundreds of milligrams and positive effects on human health [
activity observed at the initiation, promotion, and progression stages of cancer is considered to be a
most promising biological phenomenon [ ]. Currently, there is high interest in direct use of flavonoids
1]. Their anticancer
2,3
as anticancer drugs [3,4] or as enhancers for existing anticancer therapies [5,6].
Hops, the female inflorescences of Humulus lupulus, are a natural source of numerous compounds
that exhibit a remarkably broad spectrum of biological activities [
7–9]. One of the best known
compounds isolated from hops is 30-[3,3-dimethylallyl]-20,40,4-trihydroxy-60-methoxy- chalcone
Molecules 2018, 23, 776; doi:10.3390/molecules23040776
www.mdpi.com/journal/molecules

Molecules 2018, 23, 776
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(xanthohumol,
central nervous systems modulation, antimicrobial, anti-parasite, anti-obesity, hepatic protection and
antioxidant effects [ 10]. Apart from xanthohumol, many other xanthohumol derivatives, abbreviated
with capital letters from B to M, have been identified in hops so far [ 11 13]. Little is known however
about their biological activities [13 17], mainly due to the fact that only trace amounts of these
compounds are found in the plant material. Xanthohumol0s extraordinary biological properties have
given rise to a couple of reports concerning chemical [13 20] and microbial [21 23] modifications of
1), which exhibits various biological properties such as anticancer, anti-inflammatory,
7–
8,
–
–
–
–
these compounds that might increase its potency. Few of them, however, relate to the synthesis and
biological activity [15,17].
While there is a great interest in developing new synthetic flavonoid-like compounds,
the biological potential of many of the known flavonoids remains unevaluated. This is especially
important with respect to the fact that these compounds can be, or already are, a part of our diet.
Recently, a few reports have stated that chalcones containing a dimethylchromene moiety can be
more effective anti-inflammatory agents than the common anti-inflammatory drug indometacin [24
]
or exhibit lower cytotoxicity towards normal cell lines than non-cyclized derivatives [20].
Furthermore, some modified chalcones with cyclized prenyl groups exhibit strong antiproliferative
activity in vitro and in vivo [25,26].
Our previous report concerning the antiproliferative activity of xanthohumol microbial
metabolites revealed that
α
,
β-dihydroxanthohumol, which is found in trace amounts in hops,
was more active than xanthohumol (1) [23]. This finding led us to investigate biological activity
of other xanthohumol compounds, along with an assessment of their structure-activity relationships.
Herein we wish to report the synthesis and biological evaluation of a series of xanthohumol-derived
compounds with cyclized prenyl groups. Moreover, we would like to present some interesting
synthetic procedures allowing direct and regioselective cyclisation of prenyl groups that have not yet
been reported.
2. Results and Discussion
Previous reports on the synthesis of minor hops prenylchalconoids by Vogel and Heilmann [15
suggested that there were only slight differences among xanthohumol derivatives when comparing
their cytotoxic activity against HeLa cells. However, cyclized xanthohumol C ( ) has better activity
than its 100,200-dihydroderivative
15]. Because the purpose of our research was to obtain derivatives
of xanthohumol ( ) with a cyclic prenyl group, we isolated our primary compound from spent hops,
which is a rich source and it can be easily purified in considerable amounts [27].
]
2
3
[
1
1
Modifying the procedure described by Vogel and Heilmann [15] for the reaction of
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in anhydrous 1,4-dioxane at 95 C we obtained
1 and
◦
2
in 74% yield (Scheme 1). By application of straightforward chemistry, without protection of
hydroxyl groups, we were expecting to observe traces of other cyclization products, since in
1
there are two adjacent hydroxyl groups that could undergo cyclization to a dimethylchromene moiety.
However, no other cyclisation products were detected in the reaction mixture, which is in agreement
with other reports concerning DDQ cyclisation of prenyl groups and reaffirms the selectivity of the
reaction [28].
00 00
1 ,2 -Dihydroxanthohumol C (3) was obtained using the modified method described by Nerander
and Papi Reddy [29]. Xanthohumol (1) was stirred with anhydrous aluminum chloride in methylene
chloride (DCM) for 5 h at room temperature to afford in a yield of 87% (Scheme 1). It is noteworthy
that in this reaction no side products (for example ) were formed. The modification associated with
3
4
the use of an anhydrous AlCl3, instead of the boron trifluoride diethyl etherate, was due to strong
complexation of boron trifluoride, which caused problems for its removal, and reduced the final yield
of
characterized by low yields of
explained by a possible complexation of the hydroxyl group at C-20 and the carbonyl oxygen atom
4
(42–53% yield). In turn reactions with other Lewis acids, like ZnCl2, SnCl2, SnCl4, PdCl2 also were
3
and did not lead to formation of product . This phenomenon can be
4

Molecules 2018, 23, 776
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preventing other side products from being formed, not only prenyl group cyclisation, but also the
flavanone compound—isoxanthohumol (5).
Since our investigation focused on other new prenyl group cyclisation products
4 or 6 and at
this point we did not find literature data concerning methods allowing to change the regioselectivity
of cyclisation, therefor◦e we performed series of reactions in which reagent amounts (0.3–3.0 equiv.),
◦
◦
temperatures (0 C, 20 C, 50 C) and solvents (THF, DCM, DMF, DMSO) were modified. These efforts
also failed to provide any detectable amounts of the desired compounds. In the next approach we tried
to selectively block the phenolic groups at C-4 and C-40, leaving only one possible site of cyclisation.
The protection was achieved using methoxymethyl chloride (MOMCl) with K2CO3 in acetone to
give
noting that even with an elevated molar ratio of MOMCl the only product formed by this method
was . Another method involving N,N-diisopropylethylamine led to mixtures, and finally the fully
7 (Scheme 1) by a slow and controlled addition of MOMCl monitored by HPLC. It is worth
7
protected compound.
Scheme 1. General synthesis scheme of xanthohumol derived compounds. Reagents and conditions:
◦
(a) DDQ, 1,4 dioxane, 95 C (b) AlCl3, CH2Cl2, rt; (c) MOMCl, K2CO3, acetone, rt; (d) TFA, CH2Cl2, rt;
◦
◦
(e) H2, 5% Pd/C, MeOH, rt; (f) aq 1% KOH, 0 C; (g) AlCl3, MeCN, 50 C; (h) aq 1% piperidine reflux (i)
◦
I2, pyridine, 90 C; (j) anhydrous AlCl3, MeCN, rt.

Molecules 2018, 23, 776
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Interestingly, the subsequent oxidative cyclisation of
expected protected derivative of
However, deprotection with trifluoroacetic acid (TFA) in DCM led to another interesting product.
Purification and spectroscopic analysis proved it was , instead of . This encouraged us to perform
a series of reactions to test this catalyst with . The reaction of with TFA (1% v/v) in DCM
at room temperature afforded mainly (71%), along with a small amount of (18%) (Scheme 1).
7
with DDQ failed to give the
6, even when increased amounts of DDQ were applied.
4
6
1
1
4
3
Noteworthy, so far there are no reports concerning direct cyclisation of the prenyl moiety to the C-20
hydroxyl group in the presence of a free hydroxyl group at C-40, the synthetic method leading from
1
to 4. Therefore, we carried out a few experiments to test the preliminary conditions and to find an
explanation for such a phenomenon (Table 1).
Table 1. Cyclisation of xanthohumol (1) with TFA under different reaction conditions 1.
Temperature 2
TFA Amount 3
◦
4
4
4
4
4
4
C
%
of 1
%
of 3
%
of 4
4/3
% (v/v)
%
of 1
%
of 3
%
of 4
4/3
0
22
39
54.6
16.7
0
9.5
16.7
18.7
35.9
66.6
81.3
3.8
4.0
4.3
0.1
0.5
1
97.5
46.5
10.3
0.4
9.8
16.8
2.1
43.7
72.9
5.3
4.5
4.3
1
3
2
The reaction conditions are specified in the Materials and Methods section. Reactions with 0.8 % (v/v) of TFA.
4
Reactions performed at room temperature. Conversions determined by HPLC.
The results presented in Table 1 show that an increased temperature and a decreased amount of
TFA favor cyclisation to over . Obviously, increasing the temperature and TFA amount improved
4
3
the reaction yield, as well. The proposed mechanism is shown in Scheme 2. This mechanism
proposal is also supported by the fact, that by using the same procedure with other organic
acids (formic acid, acetic acid, propionic acid, oxalic acid, maleic acid and picric acid) added in 1%
(v/v) or molar equivalent to 1% of TFA did not lead to compound
Andrade-Carrera et al. [28]. Only in case of trichloroacetic acid and prolonged reaction times were
traces of found. Reactions with inorganic acids (H2SO4, H3PO4) led to isomerisation of to with
4, what was also observed by
4
1
5
formation of many other side products. Presumably, under anhydrous conditions TFA acts as an
acid-base catalyst allowing intramolecular hydrogen transfer (Scheme 2). Thus, the first step involves
protonation of the carbonyl group by TFA and further deprotonation of one of hydroxyl groups in ring
A leading to the formation of a quinone-like intermediate. Then, protonation of the double bond with
0
0
a hydrogen from a hydroxyl group (C-2 or C-4 ), formation of the tertiary carbocation and cyclisation
with a nucleophilic oxygen followed by a quinone to phenol rearrangement supported by TFA via
protonation and deprotonation. Possible hydrogen bond formed with an adjacent enol might stabilize
the oxygen anion at C-20, what might hinder further protonation by TFA and may be responsible for
the regioselectivity of the proposed mechanism. Nevertheless, more efforts have to be made to clarify
and understand this unusual cyclisation. It should be noted that the only report found describing
similar prenyl group cyclisation are studies of 6-prenylnaringenin by Mizobuchi who applied TFA in
chloroform as a method of unequivocal differentiation between 6- and 8-prenylnaringenin, but without
any comments or further investigation however [30].
Since compound
4 can be easily prepared, we decided to obtain 6 by dehydrogenation of the
dimethylchromane ring. Thus we employed several dimethylchromane ring dehydrogenation methods
using SeO2 and DDQ, but only the reaction with excess of DDQ in benzene or 1,4-dioxane resulted in
formation of 6. A preparative scale dehydrogenation reaction was conducted using the same method
as for the synthesis of 2 (Scheme 1) and it provided 6 in a yield of 17%.
Given the results of our previous studies indicating that antiproliferative activity of
the xanthohumol microbial metabolite
xanthohumol, [23] we decided to evaluate the impact of the
antiproliferative activity of -dihydrochalcone derivatives of
α
,
β
-dihydroxanthohumol was comparable to that of
-unsaturated bond on the
and Thus, and were
α,β
3
α
,β
4.
3
4

Molecules 2018, 23, 776
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hydrogenated using a previously described method [3100] with 5% palladium on charcoal in methanol
00 00
00
α
to afford 1 ,2 ,
α
,
β
-tetrahydroxanthohumol C (
8
) and 1 ,2 ,
,β-tetrahydroxanthohumol K (9) in good
yields (91% and 88%, respectively, Scheme 1).
00 00
Scheme 2. Proposed mechanism of cyclisation of xanthohumol (1) to 1 ,2 -dihydroxanthohumol K (4)
00 00
and 1 ,2 -dihydroxanthohumol C (3) by TFA.
In order to study the effect of cyclization on the antiproliferative activity of different classes
of flavonoids (flavanones and flavones) we also obtained isoxanthohumol ( ) by the base catalysed
isomerisation of xanthohumol ( ) using the procedure previously reported by Bartman´ska et al. [27
(Scheme 1). Subsequently application of reaction conditions similar to those for the synthesis of
afforded 100,200-dihydroisoxanthohumol C (10) in 76% yield from isoxanthohumol (
5
1
]
3
5) as a substrate
(Scheme 1). Modification of the reaction conditions involving the use of acetonitrile, increased
temperature and extended reaction time resulted in cyclization followed by demethylation of 10
to give 11a in 80% yield. Interestingly, TLC and HPLC analysis of the reaction mixture indicated the
formation of only one product, but further NMR analysis clearly indicated that the product obtained
after chromatographic purification on silica gel is a mixture of two compounds 11a and 11b (Scheme 3).
The analytical methods applied did not unambiguously determine the 11a and 11b ratio in the mixture.
Nevertheless, such determination would probably not be useful, since literature data presented by
Simmler et al. [32] indicates that chalcone-flavanone isomerization leading to isomerization between
11a and 11b is inevitable during biological assays like in vitro cytotoxicity tests, therefore we decided
to use the mixture of compounds 11a and 11b in the subsequent antiproliferative activity assay.
Scheme 3. Chalcone-flavanone isomerization of 11a to 11b.
The next step on our synthetic pathway was to obtain a product with a dimethylchromene
moiety from isoxanthohumol (5), thus cyclization with DDQ was the method of choice. In order
to obtain isoxanthohumol C, a number of reactions were performed with different DDQ amounts
(0.3–1.2 equiv.) temperatures (0–100 ◦C) and addition of DDQ over time, but in all cases cyclization
of the prenyl group occurred simultaneously with dehydrogenation of C2-C3 bond resulting in
2,3-dehydroisoxanthohumol C (12). A reaction set up for the complete conversion of
5 resulted
in 12 in 76% yield (Scheme 1). Another synthetic approach was to isomerize xanthohumol C (
2
)
to isoxanthohumol C. Reactions with sodium and potassium hydroxides or with sodium acetate
in ethanol [33] did not lead to the formation of desired product. Even prolonged reaction times
(over 2 days), resulted in mixtures of many side products, therefore, we applied organic bases as
catalysts, as such procedures in some cases seems to be more effective [34
,35]. Reactions in water
with addition of 2% (v/v) diethylamine, diisopropylamine, triethylamine, pyridine, pyrrolidine and

Molecules 2018, 23, 776
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piperidine carried out at room temperature for up to 48 h did not result in any flavanone compounds.
In the reaction with piperidine small amounts of the unexpected xanthohumol K (
6) were found
(18% conversion, Scheme 1).
Further reaction under reflux conditions yielded xanthohumol K (
reaction was rather unexpected we decided to test it further with xanthohumol (
), 100,200-dihydroxanthohumol C ( ) and 100,200-dihydroxanthohumol K (
). We thus obtained
isoxanthohumol ( ) from xanthohumol ( ), and xanthohumol C ( ) from xanthohumol K ( ), whereas in
case of and there were no reaction products. These results indicate the significance of the
double bond of the dimethylchromene moiety for the piperidine-catalyzed isomerization between
and . Furthermore it also indicates that compounds and are stable under aqueous base
3
) in 66% yield. Since such a
1), xanthohumol K
(6
3
4
5
1
2
6
3
4
2
6
2
3
conditions and therefore possibly less prone to chalcone-flavanone isomerisation during in vitro
assays. Interestingly, to the best of our knowledge there are no reports of such isomerizations of
chromenones, therefore it is highly possible that such an isomerization is related only to chromenes
with a carbonyl group in a position ortho to one of the reactive hydroxyl groups. Although this unusual
isomerization requires more explanation, we would like to propose the reaction mechanism shown
in Scheme 4 based on our observations. The first step involves deprotonation of hydroxyl groups
and rearrangement of aromatic and chromene ring
π-bonds resulting in an electrocyclic chromene
ring opening. Subsequent rearrangement of the -bond system, stabilized by the adjacent carbonyl
π
group, enables prenyl group isomerization and further electrocyclic new chromene ring formation.
Therefore the reaction seems to be an anionic variant of an oxy-Cope rearrangement, however further
investigation is required to confirm the role of the carbonyl group and conjugated
bond-chalcone ring B system.
α,β-unsaturated
Scheme 4. Proposed isomerization mechanism for the conversion of xanthohumol C (
xanthohumol K (6).
2) into
One of the commonly observed structure-activity relationships among flavonoids is the
importance of the C2-C3 double bond positively influencing the biological activity of flavonoids with a
heterocyclic ring C [36–38]. Although, no prenyl or geranyl flavones are detected in hops we decided to
obtain 2,3-dehydroisoxanthohumol (13) and determine its antiproliferative activity. Among methods
of direct oxidative cyclization of chalcones to flavones the most promising method is the reaction of
the chalcone with iodine in DMSO [39]. However, such a reaction with 1, despite the wide range
of iodine amounts added to the reaction mixture (0.01–1 equiv.) resulted in a complex mixture of
0
many chalcones and flavones, probably with iodine substituted carbons at C-5 or prenyl group iodine
addition reaction products. Therefore, another approach was to start from isoxanthohumol (
an iodine-pyridine complex that should not lead to unwanted substitutions or additions. Reaction of
with I2-pyridine afforded 13 in 60% yield. Following our synthetic route for cyclized products we
5) and use
5
performed the reaction of 13 with AlCl3 in acetonitrile, because of the very limited solubility of 13 in
DCM. The reaction gave 14 in 69% yield.

Molecules 2018, 23, 776
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To compare the activity of compounds obtained here with our previous results concerning the
antiproliferative properties of prenylated hops flavonoids and their fungal metabolites, we decided to
use the same antiproliferative assay and the same three cancer cell lines: prostate (PC-3), colon (HT-29)
and breast cancer (MCF-7) [23,40].
Antiproliferative potency was evaluated using the SRB assay and expressed as IC50 values, which
are the concentration of a compound that inhibits the proliferation rate of tumor cells by 50% as
compared to untreated control cells [41]. Four chalcones (
), three flavanones (10 11a 11b) and three flavones (12
Xanthohumol ( ) and cisplatin were used as reference compounds. The results are presented in Table 2.
All tested compounds exhibited moderate or strong antiproliferative activity wherein the most active
was xanthohumol ( ). The MCF-7 cell line is was the most vulnerable among the tested cell lines,
2–
4,
6), two
α,β-dihydrochalcone derivatives
(8
,
9
,
,
–14) have been subjected to this assay.
1
1
which is in accordance with our previous reports [23,40].
Table 2. In vitro antiproliferative activity of xanthohumol (1) and synthetized compounds against
human cancer cell lines.
Cancer Cell Line, IC50 (µM)
Compound
PC-3
HT-29
MCF-7
1
2
3
7.0 ± 1.5
10.1 ± 3.0
49.6 ± 6.6
10.1 ± 1.1
8.1 ± 0.8
15.0 ± 1.8
15.9 ± 3.6
10.6 ± 0.6
16.7 ± 6.9
4
10.7 ± 5.9
12.5 ± 1.5
9.0 ± 6.4
6
8
9
74.9 ± 25.6
59.9 ± 5.4
68.0 ± 14.2
87.5 ± 3.2
62.7 ± 13.2
89.1 ± 3.2
83.8 ± 14.6
16.2 ± 2.1
36.0 ± 3.6
10
11a/11b
12
51.3 ± 21.2
48.9 ± 7.8
202.0 ± 0.0
85.4 ± 2.1
25.8 ± 2.9
35.3 ± 6.6
13.2 ± 3.7
120.1 ± 3.6
217.1
±
40.9
13
14
13.8 ± 1.4
20.1 ± 1.5
7.9 ± 0.5
64.6 ± 10.7
91.2 ± 17.2
9.10 ± 1.13
19.3 ± 6.1
cisplatin (reference)
12.33 ± 2.77
8.27 ± 2.10

Molecules 2018, 23, 776
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Xanthohumol C (
2
) and 100,200-dihydroxanthohumol K (
4
) exhibited the strongest activity among
the minor hop chalconoids, moreover, both of them are as active as cisplatin with respect to the PC-3
cell line. In contrast to xanthohumol ( ), xanthohumols C ( ) and K ( ) were not the subject of a broader
study of their anticancer activity. Studies by Miranda et al. have shown that was slightly less active
than against MCF-7 and ovarian cancer cells A-278 [42]. While the antiproliferative activity of in
M, after 2 days of exposure),
1
2
6
2
1
2
relation to the MCF-7 line was comparable to our results (MIC50 = 15.7
the activity in relation to the HT-29 line was much weaker.
µ
Xanthohumol C (2) has been also reported to suppress cancer invasion in relation to matrix
metalloproteinase (MMP-2 and MMP-9) expression in HT-1080 human fibrosarcoma cells and inhibited
angiogenesis by suppression of vascular endothelial growth factor (VEGF) [43]. This compound was
proven to be the strongest agent promoting neurogenesis, neuroregeneration and neuroprotection
among the seven tested hop flavonoids (
It is noteworthy that saturation of
by Vogel and Heilmann [15]. On the other hand, the saturation of the 100,200-double bond in
the opposite effect and increased the antiproliferative activity. Saturation of the -double bond in
chalcones and resulted in a significant loss of the activity (except for the MCF-7 line and compound
) indicating that the increased activity of -dihydroxanthohumol compared to xanthohumol ( ),
which was previously reported [23], is the exception rather than a rule.
Among the compounds obtained from xanthohumol ( ), but not found in hops, the most active
1
2
,
2
,
3
,
5
, isoxanthohumol C, 6- and 8-prenylnaringenin) [44].
decreased the activity, coinciding with the results obtained
had
6
α,β
3
4
8
α
,β
1
1
turned out to be 2,3-dehydroisoxanthohumol (13), which antiproliferative activities against PC-3 and
MCF-7 lines were as high as cisplatin and against HT-29 line about twice lower than that of cisplatin.
Comparing the results obtained with compounds 12, 13 and 14 it can be assumed that the free prenyl
group determines the high antiproliferative activity of the flavones tested.
3. Materials and Methods
3.1. General Information
All chemical reagents and organic solvents used for chemical synthesis were purchased from
Merck (Darmstadt, Germany), Sigma-Aldrich, (Darmstadt, Germany) or Fluka (Darmstadt, Germany)
TLC analysis was carried out on Merck silica gel 60, F254 (0.2 mm thick) plates with
chloroform/methanol (9:1 v/v) or hexane/acetone (1:1 v/v) as eluents. HPLC was performed on
a 2695 Alliance instrument (Waters, Milford, MA, USA) equipped with a Waters 2996 photodiode
.
array detector using a Cosmosil Cholester 5
µ
m (4.6
×
250 mm) (Nacalai Tesque Inc. Kyoto, Japan),
analytical HPLC column at a flow rate of 1 mL/min. A linear solvent gradient from 45% to 95% aq
MeOH containing 0.05% HCOOH over 39 min was used. The products of reactions were separated by
column chromatography on silica gel 60 (230–400 mesh, Merck) using chloroform, methanol, hexane or
acetone mixtures as eluents. Quantitative analysis of TFA reactions was performed by means of HPLC
(detection at 340 nm). NMR spectra (1H-NMR, 13C-NMR, COSY, HMQC, HMBC) were recorded on a
DRX Avance™ 600 (600 MHz) instrument (Bruker, Billerica, MA, USA) in acetone-d6, methanol-d4 or
CDCl3. UV spectra were recorded on a Cintra 303 GBC spectrophotometer (GBC Scientific Equipment,
Braeside, VIC, Australia) in methanol. Negative-ion HR-ESI-MS spectra were taken on a Bruker
microTOF-Q instrument (Bruker, Billerica, MA, USA).
3.2. Antiproliferative Assay in Vitro
3.2.1. Cells
The following established in vitro cancer cell lines were applied: MCF-7 (human breast carcinoma),
PC-3 (human prostate cancer) and HT-29 (human colon cancer). All cancer cell lines were obtained
from American Type Culture Collection (Rockville, MD, USA) and are being maintained in the Institute
of Immunology and Experimental Therapy (Wroclaw, Poland). MCF-7 cells were cultured in Eagle

Molecules 2018, 23, 776
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medium supplemented with 2 mM L-glutamine and 10 mM sodium pyruvate, 10% fetal bovine serum
and 0.8 mg/L of insulin (from Sigma–Aldrich Chemie GmbH, Steinheim, Germany). PC-3 cells were
cultured in RPMI 1640 medium, supplemented with 2 mM L-glutamine, 10% fetal bovine serum
(both from Sigma–Aldrich Chemie GmbH). HT-29 cells were cultured in mixture of OptiMEM and
RPMI 1640 (1:1) medium (purchased from Gibco, Scotland, UK), supplemented with 2 mM L-glutamine
(Sigma-Aldrich Chemie GmbH), 5% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA USA)
medium was additionally supplemented with 1 mM sodium pyruvate (Sigma-Aldrich Chemie GmbH).
All culture media were also supplemented with antibiotics: 100 units/mL penicillin and 100 µg/mL
streptomycin (purchased from Polfa Tarchomin SA, Warsaw, Poland). All cell lines were grown at
37 ◦C with 5% CO2 humidified atmosphere. Twenty-four hours before addition of tested agents,
the cells were plated in 96-well plates (Sarstedt, Nümbrecht, Germany) at a density of 104 cells
per well in 100 µL of culture medium. An assay was performed after 72 h of exposure to varying
concentrations of the tested agents (from 0.1 to 100 µg/mL). The results were calculated as the IC50
(inhibitory concentration 50%), the concentration of tested agent which inhibits 50% of the proliferation
of the cancer cell population. IC50 values were calculated for each experiment separately and mean
values
±
SD are presented in the Table 2. Each compound at each concentration was tested in triplicate
in a single experiment, which was repeated 3–5 times. Dimethyl sulphoxide, which was used as a
solvent (in a dilution corresponding to its highest concentration applied to the tested compounds),
did not exert any inhibitory effect on cell proliferation. To evaluate antiproliferative effect of tested
compounds, the SRB method was applied.
3.2.2. SRB Assay
The details of this technique were described by Skehan et al. [41]. The cells attached to the plastic
were fixed by gently layering cold 50% trichloroacetic acid (TCA) (Sigma-Aldrich Chemie GmbH)
on the top of the culture medium in each well. The plates were incubated at 4 ◦C for 1 h and then
washed five times with tap water. The background optical density was measured in the wells filled
with culture medium, without the cells. The cellular material fixed with TCA was stained with 0.4%
sulforhodamine B (Sigma-Aldrich Chemie GmbH), dissolved in 1% acetic acid (POCh, Gliwice, Poland)
for 30 min. Unbound dye was removed by rinsing (five times) with 1% acetic acid. The protein-bound
dye was extracted with 150 µL of 10 mM unbuffered Tris base (Sigma-Aldrich Chemie GmbH) for
determination of optical density (at 545 nm) in a computer-interfaced, 96-well microtiter plate reader
(Synergy H4 Hybrid Reader, Biotek Instruments, Highland Park, VT, USA).
3.3. Chemical Synthesis
3.3.1. Xanthohumol (1)
Xanthohumol (1) was isolated from supercritical carbon dioxide extracted hops (”Marynka”,
crop 2012), obtained from Production of Hop Extracts (New Chemical Syntheses Institute, Puławy,
Poland), following the method described by Bartman´ska et al. [27]. Spectroscopic data of the isolated
xanthohumol (1) were in agreement with literature values.
3.3.2. Xanthohumol C (2)
DDQ (227 mg, 1 mmol) and
1
(354 mg, 1 mmol) were dissolved in anhydrous 1,4-dioxane (25 mL).
◦
The reaction mixture was heated to 95 C for 4 h, then cooled to room temperature and 40 mL of ethyl
acetate was added. The mixture was washed with equal volumes (20 mL) of water, saturated NaHCO3
solution, water and brine. The organic phase was dried over MgSO4, evaporated and purified by
column chromatography (eluent: chloroform-methanol 9:1) to give xanthohumol C (
2
): yield 74%,
233, 285, 370 nm; 1H-NMR (600 MHz, acetone-d )
δ:
6
orange amorphous powder; UV (MeOH)
λ
max
1.42 (6H, s, H-400, H-500), 3.97 (3H, s, C60-OCH3), 5.53 (1H, d, J = 9.9 Hz, H-200), 6.00 (1H, s, H-50),
6.62 (1H, d, J = 9.9 Hz, H-100), 6.92 (2H, m, J = 7,1 Hz, H-3, H-5), 7.59 (2H, m, J = 7.1 Hz, H-2, H-6), 7.76

Molecules 2018, 23, 776
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(1H, d, J = 15.4 Hz, H-β), 7.85 (1H, d, J = 15.4 Hz, H-α ,
), and 14.85 (s, C20-OH); 13C-NMR (150 MHz
acetone-d6) δ
: 28.52 (C-400, C-500), 56.46 (C60-OCH3), 78.73 (C-300), 92.34 (C-50), 103.36 (C-30), 106.46
(C-10), 116.53 (C-100), 116.76 (C-3, C-5), 124.96 (H-0α), 126.20 (C-200), 127.87 (C-1), 131.30 (C-2, C-6),
0 0
143.71 (H-β), 160.66 (C-4), 161.01 (C-4 ), 163.24 (C-2 ), 163.64 (C-6 ), and 193.32 (C=O). HR ESI-MS m/z:
351.1234 [M − H]− (calcd for C21H20O5 − H, 351.1238).
3.3.3. 100,200-Dihydroxanthohumol C (3)
A mixture of
(100 mL) was stirred at room temperature for 5 h. The reaction mixture was filtered, the filtrate washed
with water (3 70 mL), dried over MgSO4, evaporated and purified by column chromatography (eluent:
1 (354 mg, 1 mmol), anhydrous AlCl3 (532 mg, 4 mmol) and methylene chloride
×
chloroform-methanol 9:1) to yield 100,200-dihydroxanthohumol C (
3): yield 87%, orange amorphous
powder; UV (MeOH) λmax 370 nm; 1H-NMR (600 MHz, acetone-d6) δ: 1.34 (6H, s, H-400, H-500), 1.81 (2H,
t, J = 6.8 Hz, H-200), 2.57 (2H, t, J = 6.8 Hz, H-100), 3.95 (3H, s, C60-OCH3), 5,93 (1H, s, H-50), 6.92 (2H,
m, J = 8.6 Hz, H-3, H-5), 7.60 (2H, m, J = 8.6 Hz, H-2, H-6), 7.76 (1H, d, J = 15.5 Hz, H-
J = 15.5 Hz, H-
) and 14.93 (s, C20-OH); 13C-NMR (600 MHz, acetone-d6) : 16.77 (C-100), 26.90 (C-400,
C-500), 32.60 (C-200), 56.22 (C60-OCH3), 76.68 (C-300), 92.59 (C-50), 102.47 (C-30), 105.97 (C-10), 116.79 (C-3,
α), 7.91 (1H, d,
β
δ
C-5), 125.26 (C-α), 128.07 (C-1), 131.25 (C-2, C-6), 143.31 (C-β
), 160.60 (C-40), 161.72 (C-20), 161.75 (C-60),
166.41 (C-4), 193.16 (C=O); HR ESI-MS m/z: 353.1391 [M − H]− (calcd for C21H22O5 − H, 353.1394).
3.3.4. 100,200-Dihydroxanthohumol K (4)
To a solution of
1 (354 mg, 1 mmol) in 99 mL of methylene chloride 1 mL of TFA
was added dropwise (the addition over 5 min) and the reaction mixture was stirred at room
temperature for 5 h. The reaction was stopped by a slow addition of saturated NaHCO3 while
stirring the reaction mixture vigorously, until its color changed to yellow. Then the organic phase
was washed with water (3
chromatography (eluent: chloroform-methanol 9:1) to afford 100,200-dihydroxanthohumol K (
71%, orange amorphous powder; UV (MeOH) : 1.22 (6H,
max 331 nm; 1H-NMR (600 MHz, acetone-d6)
s, H-400, H-500), 1.75 (2H, t, J = 6.7 Hz, H-200), 2.63 (2H, t, J = 6.7 Hz, H-100), 3.62 (3H, s, C60-OCH3), 6.19
(1H, s, H-50), 6.77 (1H, d, J = 16.0 Hz, H-
), 6.88 (2H, m, J = 8.6 Hz, H-3, H-5), 7.23 (1H, d, J = 16.0 Hz,
H-
), 7.48 (2H, m, J = 8.6 Hz, H-2, H-6); 13C-NMR (150 MHz, acetone-d6) : 17.39 (C-100), 26.80 (C-400,
C500), 32.61 (C-200), 55.87 (C60-OCH3), 75.07 (C-300), 92.01 (C-50), 101.98 (C-30), 111.52 (C-10), 116.72 (C-3,
×
70 mL), dried over MgSO4, evaporated and purified by column
4): yield
λ
δ
α
β
δ
C-5), 127.52 (C-1), 127.62 (C-α), 130.82 (C-2, C-6), 144.26 (C-β
), 153.72 (C-20), 157.41 (C-60), 157.56 (C-40),
160.42 (C-4), 194.46 (C=O); HR ESI-MS m/z: 353.1392 [M − H]− (calcd for C21H22O5 − H, 353.1394).
3.3.5. Isoxanthohumol (5)
Isoxanthohumol (5) was obtained from 1 by base catalyzed isomerization following the method
described by Bartman´ska et al. [27]. Spectroscopic data of the obtained isoxanthohumol (
5) were in
agreement with literature values.
3.3.6. Xanthohumol K (6)
DDQ (45.4mg, 0.2 mmol) and
4
(70.8 mg,◦0.2 mmol) were dissolved in anhydrous 1,4-dioxane
(25 mL). The reaction mixture was heated to 95 C for 4 h, then cooled to room temperature and 40 mL
of ethyl acetate was added. The reaction mixture was washed with equal volumes (20 mL) of water,
NaHCO3, water and brine. The organic phase was dried over MgSO4, evaporated and subjected to
column chromatography (eluent: hexane-acetone 1:1) to give xanthohumol K (
yellow-orange amorphous powder; UV (MeOH)
max 288, 331 nm; 1H-NMR (600 MHz, acetone-d6)
: 1.31 (6H, s, H-400, H-500), 3.67 (3H, s, C60-OCH3), 5.52 (1H, d, J = 9.9 Hz, H-200), 6.17 (1H, s, H-50),
6.64 (1H, d, J = 9.9 Hz, H-100), 6.77 (1H, d, J = 16.0 Hz, H-
), 6.88 (2H, m, J = 8.6 Hz, H-3, H-5), 7.26
(1H, d, J = 16.0 Hz, H- : 28.03
), 7.52 (2H, m, J = 8.6 Hz, H-2,H-6); 13C-NMR (150 MHz, acetone-d6)
(C-400, C-500), 55.98 (C60-OCH3), 77.12 (C-300), 92.86 (C-50), 103.92 (C-30), 112.15 (C-10), 116.80 (C-3, C-5),
6): yield 17% and 66%,
λ
δ
β
α
δ

Molecules 2018, 23, 776
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117.56 (C-100), 127.03 (C-200), 127.57 (C-1), 127.65 (H-
α
), 130.96 (C-2, C-6), 144.65 (H-
β
), 152.86 (C-20),
155.20 (C-60), 159.05 (C-40), 160.55 (C-4), 193.62 (C=O); HR ESI-MS m/z: 351.1236 [M
−
H]− (calcd for
C21H20O5 − H, 351.1238).
Alternatively, a distilled water (10 mL) piperidine was added (0.1 mL) followed by
2 (70.4 mg,
0.2 mmol). The reaction mixture was refluxed for 20 min, then cooled to room temperature, neutralized
with 1M HCl and extracted with ethyl acetate (3 x 20 mL). The organic phase was dried over MgSO4,
evaporated and subjected to column chromatography (eluent: hexane-acetone 1:1) to give compound 6
.
3.3.7. 4.40-Dimethoxymethyl xanthohumol (7)
To a mixture of
(5 mL) MOMCl (180
1
µ
(70.8 mg, 0.2 mmol) and anhydrous K2CO3 (96.6 mg, 0.35 mmol) in acetone
L, 4.6 mmol) was added dropwise at 0 ◦C. The reaction mixture was stirred
at room temperature for 3 h, then filtered and evaporated. The residue was subjected to column
chromatography using hexane-acetone 1:1 as eluent to afford the intermediate 4.40-dimethoxymethyl
1
xanthohumol (
7
): yield 85%. yellow-orange oil; UV (MeOH)
λ
max
366 nm; H-NMR: (600 MHz,
CDCl3)
δ
: 1.67 (3H, s, H-50’), 1.79 (3H, s, H-400), 3.32 (2H, d, J = 7.2 Hz, H-100), 3.48 (3H, s, MOM-CH3),
3.49 (3H, s, MOM-CH3), 3.91 (3H, s, C60-OCH3), 5.21 (2H, s, MOM-CH2), 5.22 (1H, m, H-200), 5.26 (2H,
s, MOM-CH2), 6.23 (1H, s, H-50), 7.05 (2H, m, J = 8.8 Hz, H-3, H-5), 7.54 (2H, m, J = 8.8 Hz, H-2, H-6),
7.73 (1H, d, J = 15.7 Hz, H-α), 7.79 (1H, d, J = 15.7 Hz, H-β) and 14.12 (s, C20-OH).
3.3.8. 100,200,α,β-Tetrahydroxanthohumol C (8)
A mixture of
3 (35.4 mg, 0.1 mmol) and 5% Pd/C (70.8 mg, 2 weight equiv.) in 10 mL of methanol
was stirred at room temperature. The mixture was bubbled with nitrogen for 10 min, then with
hydrogen from a balloon for another 10 min and then stirred for the next 5 min. The reaction was
filtered, evaporated and purified by column chromatography (eluent: hexane-acetone 1:1) to yield
100,200,
α,β-tetrahydroxanthohumol C (8): yield 91%, white amorphous powder; UV (MeOH) λ
max
295 nm; 1H-NMR (600 MHz, acetone-d6)
δ
: 1.32 (6H, s, H-400, H-500), 1.80 (2H, t, J = 6.8 Hz, H-200), 2.55
00
0
0
(2H, t, J = 6.8 Hz, H-1 ), 2.85 (2H, m, H-
β
), 3.26 (2H, m, H-α), 3.87 (3H, s, C6 -OCH3), 5.91 (1H, s, H-5 ),
6.76 (2H, m, J = 8.4 Hz, H-3, H-5), 7.09 (2H, m, J = 8.4 Hz, H-2, H-6), and 14.43 (s, C20-OH); 13C-NMR
(150 MHz, acetone-d6) δ β α), 56.08
: 16.71 (C-100), 26.86 (C-400, C-500), 30.78 (H- ), 32.57 (C-200), 46.69 (H-
(C6-CH3), 76.68 (C-300), 92.36 (C-50), 102.32 (C-30), 105.48 (C-10), 116.02 (C-3, C-5), 130.18 (C-2, C-6),
133.33 (C-1), 156.47 (C-4), 161.70 (C-40), 162.02 (C-60), 165.60 (C-20), and 205.55 (C=O); HR ESI-MS m/z:
355.1550 [M − H]− (calcd for C21H24O5 − H, 355.1551).
3.3.9. 100,200,α,β-Tetrahydroxanthohumol K (9)
100,200,
α
,β
-Tetrahydroxanthohumol K (
9
) was obtained according to the same procedure as
8
,
1
using 4 as a substrate. Yield 88%, white amorphous powder; UV (MeOH)
λ
max
278 nm; H-NMR
(600 MHz, acetone-d6)
δ
: 1.26 (6H, s, H-400, H-500), 1.76 (3H, t, J = 6.9 Hz, H-200), 2.60 (2H, t, J = 6.9 Hz,
), 2.91 (2H, m, H-
), and 3.65 (3H, s, C60-OCH3), 6.12 (1H, s, H-50), 6.73 (2H,
: 17.32
), 55.92 (C60-OCH3), 75.30 (C-500), 91.93
H-100), 2.82 (2H, m, H-
β
α
m, J = 8.4 Hz, H-3, H-5), 7.05 (2H, m, J = 8.4 Hz, H-2, H-6); 13C-NMR (150 MHz, acetone-d6)
δ
(C-1000), 26.80 (C-4000, C-500), 29.82 (C-
β α
), 32.63 (C-200), 47.77 (C-
0
0
(C-5 ), 102.01 (C-3 ), 113.42 (C-1 ), 115.92 (C-3, C-5), 130.04 (C-2, C-6), 133.41 (C-1), 153.15 (C-2 ), 156.34
(C-60), 156.68 (C-4), 157.46 (C-40), and 202.63 (C=O); HR ESI-MS m/z: 355.1548 [M
−
H]− (calcd for
C21H24O5 − H, 355.1551).
3.3.10. 100,200-Dihydroisoxanthohumol C (10)
A mixture of
chloride (100 mL) was stirred at room temperature for 5 h. The reaction mixture was filtered,
the filtrate washed with water (3 70 mL), dried over MgSO4, evaporated and purified by column
chromatography (eluent: chloroform-methanol 9:1) to yield 100,200-dihydroisoxanthohumol C (10):
yield 76%, beige amorphous powder; UV (MeOH) δ:
289 nm; 1H-NMR (600 MHz, acetone-d )
5 (354 mg, 1 mmol), anhydrous AlCl3 (532 mg, 4 mmol) and methylene
×
λ
max
6

Molecules 2018, 23, 776
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1.31 (3H, s, H-400), 1.33 (3H, s, H-500), 1.79 (2H, m, H-200), 2.57 (2H, m, H-100), 2.62 (1H, dd, J = 2.9 Hz,
16.2 Hz, H-3, eq), 2.93 (1H, dd, J = 12.9 Hz, 16.2 Hz, H-3, ax), 3.77 (3H, s, C5-OCH3), 5.40 (1H, dd,
J = 2.9 Hz, 12.9 Hz, H-2), 6.00 (1H, s, H-6), 6.90 (2H, m, J = 8.5 Hz, H-30, H-50), 7.40 (2H, m, J = 8.5 Hz,
H-20, H-60); 13C-NMR (600 MHz, acetone-d6) : 17.28 (C-100), 26.58 (C-400), 27.16 (C-500), 32.60 (C-200),
δ
46.09 (C-3), 55.92 (C5-OCH3), 76.33 ( C-3000), 79.56 (C-2)0, 94.62 (C-6), 102.34 (C-8), 106.10 (C-10), 116.06
0
0
0
0
(C-3 , C-5 ), 128.62 (C-2 , C-6 ), 131.48 (C-1 ), 158.34 (C-4 ), 160.95 (C-7), 161.14 (C-5), 162.54 (C-9), 188.40
(C=O); HR ESI-MS m/z: 353.1394 [M − H]− (calcd for C21H24O5 − H, 353.1394)).
3.3.11. 5,40-Dihydroxy-600,600-dimethyl-400,500-dihydropyrano-[200,300:7,8]flavanone (11a) and
5,40-dihydroxy-600,600-dimethyl-400,500-dihydropyrano-[200,300:6,7]flavanone (11b)
A mixture of
5
(177 mg, 0.5 mmol), anhydrous AlCl3 (266 mg, 2 mmol) and anhydrous
◦
acetonitrile (50 mL) was stirred at 50 C for 24 h. The reaction mixture was filtered, the filtrate
washed with water (3
×
30 mL), dried over MgSO4, evaporated and purified by column
-dihydroxy-600,600-
-dihydroxy-600,600-dimethyl-400,500-
chromatography (eluent: chloroform-methanol 9:1) to yield a mixture of 5,4
'
dimethyl-400,500-dihydropyrano-[200,300:7,8]flavanone (11a) and 5,4
'
dihydropyrano-[200,300:6,7]flavanone (11b). yield 80%, cream amorphous powder; UV (MeOH)
λ
max
1
293, 333 nm; H-NMR (600 MHz, acetone-d6)
δ
: 1.31 (3H, s, H-400), 1.33 (3H, s, H-500), 1.79 (2H, m,
H-200), 2.56 (2H, m, H-100), 2.77 (1H, dd, J = 3.0 Hz, 16.9 Hz, H-3, eq), 3.16 (1H, ddd, J = 1.36, 12.9 Hz,
16.9 Hz, H-3a, H-3b, ax), 5.50 (1H, dd, J = 3.0 Hz, 12.9 Hz, H-2), 5.83 (1H, s, H-6), 6.91 (2H, m, J = 8.5 Hz,
H-30, H-500)0, 7.42 (2H, m, J = 8.5 Hz,0H0 -20, H-60), 11.88 (s, C5-OH); 13C-NMR (600 MHz, acetone-d6)
δ:
00
00
00
16.94 (C-1 ), 26.56 (C-4 ), 27.22 (C-5 ), 32.39 (C-2 ), 43.34 (C-3a), 43.39 (C-3b), 76.82 (C-3 ), 79.87 (C-2),
97.43 (C-6a), 97.50 (C-6b), 101.51 (C-8a), 101.54 (C-8b), 103.24 (C-10a), 103.30 (C-10b), 116.13 (C-30a,
C- 50a),116.21 (C-30b, C-50b), 128.86 (C-20, C-60), 130.93 (C-10), 158.56 (C-40a), 158.66 (C- 40b), 161.10
(C-7a), 161.12 (C-7b), 162.13 (C-9a), 162.39 (C-9b), 163.53 (C-5a), 163.58 (C- 5b), 197.40 (C=O a), 197.48
(C=O b); HR ESI-MS m/z: 339.1234[M − H]− (calcd for C20H20O5 − H, 339.1238).
3.3.12. 2,3-Dehydroisoxanthohumol C (12)
DDQ (45.4 mg, 2 equiv.) and
5
(35.4 mg, 1 equiv.) were dissolved in anhydrous 1,4-dioxane
◦
(10 mL). The reaction mixture was heated to 95 C for 4 h, then cooled to room temperature and
40 mL of ethyl acetate was added. The reaction mixture was washed with equal volumes (20 mL)
of water, NaHCO3, water and brine. The organic phase was dried over MgSO4, evaporated and
subjected to column chromatography (eluent: hexane-acetone 1:1) to give 2,3-dehydroisoxanthohumol
C (12): yield 76%, yellow amorphous powder; UV (MeOH)
λ
max
238, 289, 335 nm; 1H-NMR (600 MHz,
00
00
00
acetone-d6)
δ
1.48 (6H, s, H-4 , H-5 ), 3.88 (3H, s, C5-OCH3), 5.77 (2H, d, J = 10.0 Hz, H-2 ), 6.39 (1H, s,
H-6), 6.47 (1H, s, H-3), 6.95 (2H, d, J = 10.0 Hz, H-100) 7.03 (2H, m, J = 8.8 Hz, H-30, H-50), 7.90 (2H, m,
J = 8.8 Hz, H-20, H-60); 13C-NMR (600 MHz, acetone-d6)
δ
: 28.30 (C-400, C-500), 56.49 (C5-OCH3), 78.65
(C-300), 97.41 (C-6), 103.45 (C-8), 107.45 (C-3), 109.55 (C-10), 116.75 (C-100), 116.84 (C-30, C-50), 123.64
(C-10), 128.63 (C-200), 128.64 (C-20, C-60), 154.61 (C-9), 158.46 (C-7), 160.95 (C-2), 161.32 (C-40), 161.58
(C-5), 176.48 (C=O); HR ESI-MS m/z: 349.1080 [M − H]− (calcd for C21H18O5 − H, 349.1081).
3.3.13. 2,3-Dehydroisoxanthohumol (13)
Iodine (38 mg, 0.3 mmol) was dissolved in 10 mL of anhydrous pyridine and the mixture was
stirred at room temperature, after 1 h isoxanthohumol (5) (106 mg, 0.3 mmol) was added and the
mixture was stirred at 90 ◦C for 3h. The reaction was cooled and stopped by adding 10 mL of water
and 3 mL of saturated Na2S2O3 solution, then neutralized with 1 M HCl solution and extracted with
ethyl acetate (3
×
40 mL), dried over MgSO4, evaporated and purified by column chromatography
(eluent: chloroform-methanol 5:1) to yield 2,3-dehydroisoxanthohumol (13): yield 60%, white-yellow
amorphous powder; UV (MeOH)
λ
268, 335 nm; 1H-NMR (600 MHz, metanol-d )
δ 1.67 (3H, s,
max
4
H-40000), 1.81 (3H, s, H-500), 3.54 (2H, d, J = 6.7 Hz, H-100), 3.86 (1H, s, C5-0OCH3), 5.23 (1H, t, J = 6.7 Hz
,
0
0
H-2 ), 6.43(1H, s, H-6), 6.52 (1H, s, H-3), 6.91 (2H, m, J = 8.8 Hz, H-3 , H-5 ), 7.78 (2H, m, J = 8.8 Hz, H-2 ,

Molecules 2018, 23, 776
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H-60); 13C-NMR (150 MHz, metanol-d4)
δ
: 18. 24 (C-500), 22.71 (C-100), 25.92 (C-400), 56.09 (C5-OCH3),
96.60 (C-6), 106.04 (C-3), 108.09 (C-10), 109.32 (C-8), 116.67 (C-30, C-50), 123.36 (C-10), 123.43 (C-200),
128.81 (C-20, C-60), 132.51 (C-300), 158.23 (C-9), 159.57 (C-5), 161.67 (C-7), 161.68 (C-40), 163.29 (C-2),
180.58 (C=O); HR ESI-MS m/z: 351.1237 [M − H]− (calcd for C21H20O5 − H, 351.1238).
3.3.14. 100,200-Dihydro-2,3-dehydroisoxanthohumol C (14)
A mixture of 13 (35 mg, 0.1 mmol), anhydrous AlCl3 (53 mg, 0.4 mmol) and anhydrous acetonitrile
(20 mL) was stirred at room temperature for 2 h. The reaction mixture was filtered, the filtrate washed
with water (3
×
10 mL), dried over MgSO4, evaporated and purified by column chromatography
(eluent: chloroform-methanol 5:1) to yield 100,200-dihydro-2,3-dehydroiso- xanthohumol C (14): yield
69%, yellow amorph0o0us powder; UV (MeOH)
λ
max 271, 335 nm; 1H-NMR (600 MHz, metanol-d4)
δ
:
00
00
00
1.40 (6H, s, H-4 , H-5 ), 1.95 (2H, m, H-2 ), 2.96 (2H, m, H-1 ), 3.85 (3H, s, C5-OCH3), 6.39 (1H, s, H-6),
6.59 (1H, s, H-3), 6.94 (2H, m, J = 8.8 Hz, H-30, H-50), 7.85 (2H, m, J = 8.8 Hz, H-20, H-60); 13C-NMR
(150 MHz, metanol-d4)
δ
: 17.59 (C-100), 26.86 (C-400, C-500), 32.76 (C-200), 56.35 (C5-OCH3), 77.43 (C-300),
98.33 (C-6), 103.03 (C-8), 106.33 (C-3), 108.74 (C-10), 117.04 (C-30, C-50), 123.41 (C-10), 129.07 (C-20,
0
0
C-6 ), 152.97 (C-9), 158.69 (C-5), 160.09 (C-7), 160.75 (C-4 ), 163.48 (C-2), 180.49 (C=O); HR ESI-MS m/z:
351.1239 [M − H]− (calcd for C21H20O5 − H, 351.1238).
Supplementary materials contain all obtained 1H-NMR and 13C-NMR spectra of compounds
presented in the manuscript.
3.3.15. TFA Catalyzed Cyclisation of xanthohumol (1) with Various Catalyst Concentration and at
Different Temperatures
Test reactions from Table 1 were performed in accordance to the synthesis of
Section 3.3.4. There were differences in temperature and TFA amounts, as noted in Table 1.
Also, the reactions were carried out in a smaller scale, using less substrate (35.4 mg), smaller solvent
4, described in
1
volume (10 mL) and shorter reaction time (3 h). In each experiment after three hours 10 mL of water
was added to the reaction mixture, while stirring vigorously. The organic phase was separated and
dried over MgSO4. Then 0.1 mL of the organic phase was mixed with 1 mL of HPLC grade MeOH and
analyzed by HPLC.
4. Conclusions
The results of in vitro tests indicate that chromane- and chromene-like cyclic prenylchalconoids
are potent antiproliferative agents. These observations as well as the fact that some of the compounds
obtained (
further study on the pharmacological properties of hop compounds [45]. The most promising seem
to be the minor hops prenlylchalconoids xanthohumol C ( 4)
) and 100,200-dihydroxanthohumol K (
2, 3, 4) arise as a result of metabolism in mammalian systems justify our research and
2
and non-natural 2,3-dehydroisoxanthohumol (13), which exhibited activity comparable to cisplatin.
With high probability, these compounds will not be metabolized by gastrointestinal microbiome like
xanthohumol (1) to 8-prenylnaringenin, which besides its assumed beneficial activities may promote
mammary and endometrial cancers [46]. The development of efficient regioselective xanthohumol (1)
00 00
prenyl group cyclisation reaction to give 1 ,2 -dihydroxantohumol K (
4) may be further applied in the
synthesis of other compounds with the chromane moiety.
Supplementary Materials: The following are available online. Supplementary materials contain all obtained
1H-NMR and 13C-NMR spectra of compounds presented in the manuscript.
Acknowledgments: The research was financed by National Science Centre, Poland, Grant No. 2016/21/B/
NZ7/02759. The publication was supported by the Wroclaw Centre of Biotechnology, programme The Leading
National Research Centre (KNOW) for the years 2014–2018. Special thanks to “Elk Crew—Problem of the Week”
group from University of Graz for fruitful discussions regarding reaction mechanisms.
Author Contributions: Jarosław Popłon´ski conceived the research, designed and performed synthetic part of the
research and partially wrote the manuscript. Eliza Turlej performed all biological assays. Sandra Sordon and

Molecules 2018, 23, 776
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Agnieszka Bartman´ska helped in purification and spectral analysis of obtained compounds. Tomasz Tronina
purified xanthohumol from spent hops. Joanna Wietrzyk was supervising biological assays. Ewa Huszcza partially
wrote the manuscript and supervised the progress of the research.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all compounds are available from the authors for possible research projects
in cooperation.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).