One-Pot Synthesis of Benzopyran-4-ones with Cancer Preventive and Therapeutic Potential

Article abstract of DOI:10.1002/ejoc.201501278

A one-pot synthesis of novel benzopyran-4-ones is described. In a tandem reaction, organobase-catalysed Michael addition of R¹COCH₂COR² on chromone-3-carboxylic acid led to decarboxylation and pyran-4-one ring opening of the latter. This was followed by chromone-and/or chromanone ring closure of the resulting Michael adducts when R¹ is an ortho-hydroxyaryl group. Antioxidant testing of 14 derivatives identified strong antiradical properties of chromanones 3o-r (2.1-3.1 μmol Trolox equiv./μmol compound in the DPPH assay). Chromanones 3p and 3r and 2-styrylchromone 3k were also most potent in inducing the cytoprotective Keap1-Nrf2 signalling pathway in a reporter gene assay (fivefold induction at concentrations <3 μM). Of the seven compounds evaluated for antiproliferative activities, 3k and 3r were the most active, inhibiting leukaemia K562 cell proliferation by 50 % after 72 h at concentrations of 4.5 and 7.9 μM, whereas normal peripheral blood mononuclear cells were not affected. Chromanones 3p and 3r and 2-styrylchromones 3k potently activate the Nrf2 response in the AREc32 cell line at low micromolar concentrations (C5 <3 μM). Compounds 3k and 3r are also the most active in inhibiting cell proliferation by 50 % after 72 h incubation at concentrations of 4.5 and 7.7 μM, whereas normal peripheral blood mononuclear cells were not affected.

Full text of DOI:10.1002/ejoc.201501278

DOI: 10.1002/ejoc.201501278
Full Paper
Drug Discovery
One-Pot Synthesis of Benzopyran-4-ones with Cancer
Preventive and Therapeutic Potential
Oualid Talhi*[a,b] Lidia Brodziak-Jarosz,[c,d] Jana Panning,[e,f] Barbora Orlikova,[f,g]
Clemens Zwergel,[e,h] Tzvetomira Tzanova,[h] Stéphanie Philippot,[h] Diana C. G. A. Pinto,[a]
Filipe A. Almeida Paz,[i] Clarissa Gerhäuser,[c] Tobias P. Dick,[d] Claus Jacob,[e]
Marc Diederich,[f,g] Denyse Bagrel,[h] Gilbert Kirsch,[h] and Artur M. S. Silva*[a]
Abstract: A one-pot synthesis of novel benzopyran-4-ones is Chromanones 3p and 3r and 2-styrylchromone 3k were also
described. In a tandem reaction, organobase-catalysed Michael
addition of R1COCH2COR2 on chromone-3-carboxylic acid led
to decarboxylation and pyran-4-one ring opening of the latter.
This was followed by chromone- and/or chromanone ring clo-
sure of the resulting Michael adducts when R1 is an ortho-hy-
droxyaryl group. Antioxidant testing of 14 derivatives identified
strong antiradical properties of chromanones 3o–r (2.1–
3.1 μmol Trolox equiv./μmol compound in the DPPH assay).
most potent in inducing the cytoprotective Keap1-Nrf2 signal-
ling pathway in a reporter gene assay (fivefold induction at con-
centrations <3 μM). Of the seven compounds evaluated for anti-
proliferative activities, 3k and 3r were the most active, inhibit-
ing leukaemia K562 cell proliferation by 50 % after 72 h at con-
centrations of 4.5 and 7.9 μM, whereas normal peripheral blood
mononuclear cells were not affected.
Introduction
Derivatives with a basic chromone structure are ubiquitously
found in the plant kingdom. Their antimicrobial, antiviral, anti-
cancer, anti-inflammatory, and antioxidant effects are profitable
to human health.[2] Further representatives of the benzopyran-
4-one nucleus are the chromanones, among which, several are
in use for the treatment of various ailments such as hyperten-
sion, asthma, ischemia, and urinary incontinence.[3,4] The flav-
one class of compounds represents the most abundant class of
phytochemical components of plants, fruit and vegetables.[4]
These compounds are associated with a wide range of biologi-
cal activities and nutritional benefits, and are mostly recognised
as anticancer, anti-inflammatory and antioxidant agents.[5] In
parallel, 2-styrylchromones, although scarce in nature, represent
one of the most useful chromone-based scaffolds due to their
antiviral, antitumor, antimitotic, anti-inflammatory and antioxid-
ant activities.[6,7]
Recent research has revealed a multitude of biological activities
attributed to benzopyran-4-one-based compounds, including
chromones, chromanones, flavones and 2-styrylchromones.[1]
[a] Department of Chemistry & QOPNA, University of Aveiro,
3810-193 Aveiro, Portugal
E-mail: oualid.talhi@ua.pt
artur.silva@ua.pt
https://sites.google.com/site/artursilvaua/
[b] Centre de Recherche Scientifique et Technique en Analyses
Physico-Chimiques – CRAPC,
B. P. 384, Bou-Ismail, 42004 Tipaza, Algeria
[c] Division of Epigenomics and Cancer Risk Factors, German Cancer Research
Center (DKFZ),
Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
[d] German Cancer Research Center (DKFZ), Division of Redox Regulation,
DKFZ-ZMBH Alliance,
Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
[e] Division of Bioorganic Chemistry, School of Pharmacy, University of
Saarland
Campus B2 1, 66123 Saarbrücken, Germany
[f] Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC),
Hôpital Kirchberg
9, rue Edward Steichen, 2540 Luxembourg, Luxembourg
[g] Université de Lorraine, Lab. Structure et Réactivité des Systèmes
Moléculaires Complexes, UMR 7565,
Due to the growing medicinal interest in benzopyran-4-one-
based compounds, several multistage approaches for the syn-
thesis of novel derivatives with new biological profiles and/or
enhanced bioactivities have been developed. For example, in
the last decade, several research groups have been involved in
the synthesis of 3-substituted-flavones and -2-styrylchromones
in an effort to find compounds with improved antioxidant and
antitumor activities.[7,8] Moreover, an interesting series of potent
antioxidant agents, 6-hydroxy-7-methoxy-4-chromanones and
chroman-2-carboxamides, have been described,[9] thus turning
these scaffolds more attractive for biological applications. Nev-
ertheless, better knowledge on structure-activity relationships
(SAR) is required to design benzopyran-4-one frameworks that
are suitable for testing of in vitro and in vivo biological interac-
1 Boulevard Arago, Metz, France
[h] Department of Pharmacy, College of Pharmacy, Seoul National University
1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea
[i] Department of Chemistry & CICECO, University of Aveiro,
3810-193 Aveiro, Portugal
Supporting information and ORCID(s) from the author(s) for this article
are available on the WWW under http://dx.doi.org/10.1002/
ejoc.201501278.
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tions. Accordingly, convenient synthetic routes for novel benzo- from the activation of cytoprotective mechanisms to the stimu-
pyran-4-one-based compounds are still of urgent need in the lation of death-promoting signalling.[15]
medicinal chemistry field.
The present study aimed to provide a simplified synthesis of
17 novel benzopyran-4-ones 3a–g, 3j–r and 3o′, bearing the
HOPO moiety in the 3-position of chromones, flavones and 2-
styrylchromones along with 2,3-disubstituted chromanones in-
corporating the curcuminoid-type moiety (CINA) as an impor-
tant scaffold for biological activities. To compare their biological
properties and deduce SAR correlations, the compounds were
tested for antioxidant activity using ferric ion reducing FRAP
and DPPH free radical scavenging tests. Anticarcinogenic poten-
tial was evaluated by measuring induction of cytoprotective
Keap1-Nrf2 signalling,[16] and cytostatic potential in human leu-
kemia cells.[17] The combined approach identified 2-styryl-
chromone 3k and chromanone 3r as promising novel benzo-
pyran-4-one derivatives, which are now available for further
investigation of the mechanisms underlying their biological
activities.
In 2002, Tuskaev et al. described a molecular engineering
approach to increase the antiallergic activity and decrease the
toxicity of chromones.[10] They inserted a (2-hydroxyphenyl)-3-
oxoprop-1-enyl- (HOPO) moiety as a Michael acceptor group
into the chromone structure to design chalcone-like derivatives.
Nowadays, chalcones and other natural flavonoids sharing
HOPO moieties are widely recognised as compounds of phar-
macological interest. As the best example, the chalcone xantho-
humol (Figure 1), which is extracted from hop, is one of the
most important prenylated flavonoids, offering a broad spec-
trum of biological activities, including antioxidant activity,
potentiation of nerve growth factor action, induction of
NAD(P)H:quinone oxidoreductase, inhibition of diacylglycerol
acyltransferase, and inhibition of human cytochrome p450 en-
zymes.[11] Chalcones are also cytotoxic and promote cell death
through induction of apoptosis.[12] Importantly, they also pos-
sess cancer chemopreventive properties, for example by induc-
ing the cytoprotective Nrf2 signalling pathway, which has been
suggested to be mainly due to the presence of the HOPO
group.[13] Another natural compound with a Michael acceptor
moiety is curcumin (Figure 1), which is one of the most potent
natural anticancer compounds. This polyphenol with two cinna-
moyl (CINA) substitutions shows an accentuated Michael ac-
ceptor character, which could be involved in the inhibition of
tumour development in animal models of oral, gastric, intesti-
nal, colonic, hepatic and cutaneous carcinogenesis.[14]
Results and Discussion
Chemistry
The electron-withdrawing effect of both the 3-carboxylic acid
and 4-ketone functions of 1 is useful to generate a strong elec-
trophilic site at the C-2 position. Only recently, synthetic efforts
have focussed on the Michael addition on similar C-2 electron-
deficient chromones, giving rise to novel functional polycyclic
chromones[18] and functionalized 2-hydroxybenzophenones[19]
through tandem reaction processes. Peng et al.[20] reported the
synthesis of 4-substituted 3,4-dihydrocoumarins using a 1,4-
conjugate addition/double decarboxylation cascade reaction of
a ꢀ-ketocarboxylic acid on the coumarin-3-carboxylic acid.
The reaction of chromone-3-carboxylic acid 1 with primary
and secondary amines, hydrazines, cyanoacetohydrazide,
cyanoacetamide and malononitrile in different media has
mainly led to ring transformation through pyran-4-one-ring
opening and decarboxylation.[21] In the same study, 3-
(HOPO)chromone 3a was prepared through intermolecular
dimerisation and decarboxylation of chromone-3-carboxylic
acid 1 in a sodium hydroxide solution.[21]
In the present study, the behaviour of acid 1 under organo-
base catalysis was fully studied. Initially, a catalytic amount of
4-pyrrolidinopyridine (4-PPy) as a tertiary amine organic base
Figure 1. Natural cancer preventive compounds containing a HOPO- or CINA-
pharmacophore.
These few examples indicate that the Michael acceptor func- was used to convert acid 1 into the dimeric product 3a with
tionality could be an important structural feature for the biolog- an optimal yield (67 %), involving dimerisation and decarboxy-
ical activities of curcuminoids and chalcones to protect against lation reactions. A small amount of the unsubstituted chromone
chronic degenerative diseases in animal models of carcinogene- 3aa was also formed. Compound 3a was readily isolated after
sis, cardiovascular disease and neurodegeneration. Mechanisti- recrystallisation from ethanol, whereas 3aa remained dissolved
cally, it has been postulated that at least some of the effects in ethanol and was subsequently isolated by chromatography.
exerted by HOPO and CINA compounds in biological systems Under these mild conditions, the organobase-promoted dimeri-
can be attributed to the electrophilic properties of the Michael sation of 1 gave better results than the inorganic base proce-
acceptor pharmacophore. Hence, sulfur- and nitrogen-based dure (NaOH in the procedure developed by Ibrahim[21]), regard-
cellular nucleophiles can potentially react with the electrophilic less of the longer reaction time (Table 1). Here, the use of an
ꢀ-carbon of HOPO and CINA compounds. The biological re- organobase catalyst 4-PPy (or TEA, DBU) allowed the dimerisa-
sponses of cells exposed to electrophilic compounds may range tion of 1 with higher yields (up to 67 %; Table 1, entry 1).
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Table 1. Dimerisation of chromone-3-carboxylic acid 1 under organobase ca-
talysis.
(HOPO)-2-styrylchromones 3k–n and 3o′, in a one-pot organo-
base-catalysed tandem reaction (Scheme 2, Table 2).
Entry
Solvent
Organo-
base
Temp.
[°C]
Time
[h]
Yield
[%]
1
2
3
4
5
6
CH2Cl2
CHCl3
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
4-PPy
4-PPy
DBU
TEA
pyridine
room temp.
reflux
room temp.
room temp.
room temp.
48
3
24
48
> 48
> 48
67[a,b]
51[a,b]
25[a–c]
55[a–c]
[b,c]
–
[b,c]
2,6-lutidine room temp.
–
[a] Isolated yield after recrystallisation. [b] Formation of by-product 3aa was
confirmed by analytical TLC using an authentic sample. [c] Remaining starting
material 1 was identified by TLC.
Similar to the dimeric product 3a obtained through Michael
addition of the ω-formyl-2′-hydroxyacetophenone intermediate
2a on acid 1 (Scheme 1), other 1,3-dicarbonyl reagents 2a–r
could be used as a Michael donor towards 1 to afford 3, bearing
the desired HOPO moiety. We started to study the C-2 Michael
acceptor character of 1 under the action of carbon nucleophiles
derived from the 1,3-dicarbonyl compounds 2a, 2ab and 2ac,
in the presence of a catalytic amount of 4-PPy in refluxing
chloroform. As a result, the action of ω-formyl-2′-hydroxyaceto-
phenone 2a produced the dimeric compound 3a in higher
yield (74 % compared with the dimerisation procedure of 1
which yielded 67 % of 3a). The use of different diaryl-1,3-di-
carbonyl compounds (R1COCH2COR2) sharing mainly the key
function 2-hydroxyaryl group 2a–n, synthesised by using the
Baker–Venkataraman method,[22] was found to give an addi-
tional intramolecular cyclisation event of the linked ꢀ-diketone
portion into the benzopyran-4-one ring, producing a variety of
3-(HOPO)chromones 3a–d, 3-(HOPO)flavones 3e–g, 3j and 3-
Scheme 2. Michael addition of 1,3-dicarbonyl compounds 2a–n on acid 1.
Cyclisation of 1,3-dicarbonyls (or ꢀ-diketones) 2a–n into their
corresponding benzopyran-4-one nucleus failed to occur under
the operating condition of organobase catalysis (using 4-PPy) in
the absence of acid 1 (Scheme 3). Moreover, the unsubstituted
chromone 3aa does not behave as a good Michael acceptor
when tested for this type of 1,4-conjugate addition reaction
(Scheme 3). Hence, our new synthetic method for the construc-
tion of benzopyran-4-one-based compounds combines both
the Baker–Venkataraman rearrangement with the tandem
Michael addition on chromone-3-carboxylic acid 1 (Scheme 2).
Scheme 3. Effect on the starting materials of the Michael addition of 1,3-
dicarbonyl compounds 2a–n on acid 1.
An exception was found in the reactivity of 1-(mono-, or di-
methoxy-2-hydroxyphenyl)-3-styryl-1,3-dicarbonyl compounds
2o–r, for which the Michael addition on the acid 1 led to the
selective production of novel 2,3-disubtituted chromanone-
based compounds 3o–r incorporating the curcumin-type CINA
moiety and bearing an asymmetric (C-2) centre (Scheme 4,
Table 2, entries 15–19). In the case of 2o, reaction with acid
1 leads to the formation of a minor amount of 3-(HOPO)-2-
styrylchromone 3o′ (yield 7 %, Table 2, entry 15) along with the
major product of 2,3-disubstituted chromanone 3o (yield 35 %,
Table 2, entry 16).
Scheme 1. Michael addition of 1,3-dicarbonyl compounds 2a, 2ab and 2ac
on acid 1.
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Table 2. Michael addition of 1,3-dicarbonyl 2a–r on acid 1.
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Table 2. (Continued).
[a] Isolated yield after recrystallisation. [b] The formation of chromone 3aa was confirmed by analytical TLC using an authentic sample. [c] The formation of
the dimeric product 3a was confirmed by TLC. [d] The remaining starting materials 1 and 2a–r were determined by TLC.
With the above experimental findings in hand, a global quence of lower acidity of their activated methylene protons
mechanism can be proposed for the synthesis of benzopyran- and/or a bulky structure due to their high degree of methoxy-
4-one-based compounds, which offers several advantages, but substitution, thereby hindering access of the tertiary amine 4-
also presents some limits in exceptional cases (Scheme 5). For PPy. In addition, the use of benzyl-OH-protected 1,3-dicarbonyl
instance, 1,3-dicarbonyl compounds 2h and 2i did not show compounds with free 2-hydroxyaryl failed to yield the expected
reactivity (Table 2, entries 8 and 9), which could be a conse- products (data not shown). Instead, the dimeric compound 3a
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tially close to H-2, and H-2′ to H-6′′ of the ortho-hydroxyphenyl
group; this fact is also consistent with the (E)-configuration of
the C1′=C2′ double bond of HOPO (Figure 2). The same oc-
curred for flavones 3e–g, as shown for compound 3f (Figure 2).
Scheme 4. Michael addition of 1,3-dicarbonyl 2o–r on acid 1.
Figure 2. Main NOE effects observed in the NOESY spectra of compounds 3a,
3f and 3l.
and the chromone 3aa by-product were formed, indicating that
structural hindrance might indeed play a role. 4-PPy, the most
efficient tertiary amine used, allowed the formation of only neg-
ligible quantities of the by-product chromone 3aa during the
course of the dimerisation reaction compared with the use of
DBU and TEA (Table 1).
The crystallographic studies of 3a confirmed the stable C3-
C1′ s-trans conformation of the molecule as inferred from the
NOESY spectrum. In the solid state, the very strong hydrogen
bond [dD···A = 2.546(3) Å] between the 2′′-OH and the C-3′ carb-
onyl group is clearly visible (Figure 3).
The structural characterisation of the new benzopyran-4-
one-based products was established on the basis of extensive
2D NMR studies, including HSQC, HMBC and NOESY experi-
ments and further supported by X-ray crystallographic studies
1
(fully discussion in the Supporting Information). H NMR analy-
sis was used to delineate several stereochemical features that
were present in all the synthesised polyphenolics 3a–g, 3j–n
and 3o′, namely the (E)-configuration of the C2′=C1′ double
bond [J = 15.1–15.2 Hz] of the 3-(2-hydroxyphenyl)-3-oxoprop-
1-enyl (HOPO) moiety.
Further information on the conformational and stereochemi-
cal structural features of the new benzopyran-4-one-based
compounds were gained from NOESY experiments. Compound
3a must exist in its C3-C1′ s-trans-conformation according to
Figure 3. Schematic representation of the molecular unit present in the crys-
tal structure of 3a. Non-hydrogen atoms are represented as thermal ellipsoids
drawn at the 50 % probability level; hydrogen atoms are depicted as small
spheres with arbitrary radii.
In the more complex structures, the data obtained from the
1
NOESY spectra analysis, which clearly shows that H-1′ is spa- NOESY experiment supported the H NMR spectral analysis for
Scheme 5. Proposed reaction mechanism of Michael addition of 1,3-dicarbonyl 2a–r on chromone-3-carboxylic acid 1.
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the multiple double bond conjugated systems. The C-1′-C3/ hand, the chromanone derivatives 3o–r showed strong anti-
C1′′′-C3 presents an s-trans conformation of the HOPO frag- radical properties in the range of 2.1–3.1 μmol Trolox equi-
ment in all the scaffolds 3a–g, 3j–n and 3o′ (Figure 2 presents valents/μmol compound. In a previous study, the 6-hydroxy-7-
the case of 3l).
methoxy-4-chromanone displayed a radical scavenging activity
comparable to the activity of Trolox.[9] Strong radical scaveng-
ing activity of our newly synthesised chromanones 3o–r may
derive from the addition of the CINA moiety. The degree of
methoxy substitution did not strongly influence their antiradical
capacity.
Finally, in the case of chromanones 3o–r, the NOESY spectra
did not allowed us to unequivocally assign its 3D structure,
however X-ray diffraction analysis of chromanone 3o showed
centrosymmetric triclinic P1¯ crystals with the asymmetric unit
being composed of a whole molecular unit, as depicted in Fig-
ure 4. Due to the centrosymmetric nature of the crystal, the
bulk material is formed as a racemic mixture of both R and S
enantiomers. In this context, one can immediately infer that the
employed Michael protocol under organobase catalysis does
not allow an enantioselective production of 2,3-disubstituted
chromanones 3o–r.
Figure 5. Evaluation of antiradical activity of benzopyran-4-ones 3a–g and
3k–r (DPPH assay).
Figure 4. Schematic representation of the molecular unit present in the crys-
tal structure of compound 3o. Non-hydrogen atoms are represented as ther-
mal ellipsoids drawn at the 50 % probability level; hydrogen atoms are de-
picted as small spheres with arbitrary radii.
Benzopyran-4-ones 3a–g and 3k–r Activate the
Cytoprotective Keap1-Nrf2 Pathway in AREc32 Cells
Given that benzopyran-4-ones 3a–g and 3k–r contain a Michael
acceptor centre, either in the form of a HOPO group in com-
pounds 3a–g and 3k–m or in the form of a CINA group in
chromanone derivatives 3o–r, we addressed the question of
whether they activate the Keap1-Nrf2 pathway compared to
xanthohumol (XN) and sulforaphane (SFN), which were used as
positive controls.
Biological Screening
In vitro Antioxidant Activities of 3a–g, 3k–m and 3o–r
First we evaluated the antioxidant activity of benzopyran-4-
ones 3a–g, 3k–m and 3o–r by using the ferric reducing anti-
oxidant power (FRAP) technique[23a] and DPPH methods.[23b]
To monitor the Nrf2 signalling induction, we utilised the
AREc32 cell line treated with the 14 benzopyran-4-ones 3a–
g and 3k–r and positive controls SFN and XN, all used in a
concentration range of 1.56–100 μ
Nrf2 activity in a dose-dependent manner. With an approximate
M.
[16] All compounds induced
Trolox
(6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid) was used as a reference.
Of the 14 tested compounds, all except 3e were able to re- 91-fold increase above the vehicle control, chromanone 3r was
duce ferric ions in the FRAP assay, however, with about 3.9-fold identified as the most potent inducer (see the Supporting Infor-
lower efficiency than Trolox (see the Supporting Information, mation, Figure S8). The concentrations required to increase lu-
Figure S7). In the DPPH scavenging assay, chromones 3a–3d ciferase activity by fivefold (C5 value, Table 3) in relation to
˙
and all chromanones 3o–r demonstrated DPPH scavenging ac- solvent control were in the range of 2.5–15.2 μ
tivity, whereas flavones 3e–g and 2-styrylchromones 3k–m nes 3o–r, 2.9–18 μ for 2-styrylchromones 3k–m, 4.7–32.8 μ
were inactive (Figure 5). We identified chromone 3a as the most for chromones 3a–d, and >53 μ for flavones 3e–g. Overall,
potent benzopyran-4-one derivative, with an antiradical activity chromanones were the most potent class of Nrf2 inducers.
of 3.7 0.3 μmol Trolox equivalents/μmol compound. SAR anal- Compounds 3p and 3r showed C5 values of 2.5 μ , which were
yses within the chromone class of compounds revealed that lower than the C5 values achieved with the positive control
addition of methoxy 3b–c or methyl 3d groups on the chrom- compounds XN (7.8 μ ) and SFN (4.8 μ ). Inducing activity was
M for chromano-
M
M
M
M
M
M
one moiety of 3a strongly reduced the antiradical activity. Like- accompanied by weak antiproliferative effects. Compounds 3p
wise, addition of a phenyl group on the benzopyran-4-one lead- and 3r reduced cell numbers with half-maximal inhibitory con-
ing to flavone structures 3e–g or a styryl group leading to 2- centrations (IC50) of 49.9 and 41.9 μ
M, respectively (Table 3). We
styrylchromones 3k–m lowered the activity of 3a. On the other also identified Nrf2-inducing potential for the chromanone 3o,
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Table 3. Nrf2 inducing potential of benzopyran-4-ones 3a–g and 3k–r in
AREc32 cells.
at 100 μ
M
after 72 h (IC50 7.9). Styrylchromone 3k showed a
strong cytostatic effect when used at a concentration of 10 μ
M
[a]
[b]
after 72 h (IC50 4.5 μ ).
M
Compound
C5 [μM]
Tox. IC50 [μ
M]
Number & position
of OCH3
3a
3b
3c
3d
3e
3f
3g
3k
3l
3m
3o
3p
3q
3r
12.4
20.1
32.8
4.7
40.3
0
> 100 (73)
> 100 (58)
> 100 (78)
> 100 (86)
> 100 (100)
> 100 (100)
> 100 (68)
> 100 (100)
> 100 (86)
> 100 (56)
49.9
1 (C-7)
2 (C-5,7)
0
52.9
0
> 100 (3.2)
> 100 (4.5)
2.9
18.0
10.2
3.4
2.5
15.2
2.5
3 (C-7,3′,4′)
2 (C-3′,4′)
0
2 (C-3′′, 4′′)
1 (C-4′′)
1 (C-7)
2 (C-5,7)
3 (C-7,3′′′′,4′′′′)
4 (C-5,7,3′′′′,4′′′′)
1 (C-2)
n.a.
> 100 (89)
41.9
27.5
XN
SFN
7.8
4.8
44.5
[a] C5 value, concentration causing a fivefold induction of luciferase activity
(= Nrf2 activity). Values in parentheses indicate the fold induction vs. solvent
control at the indicated concentration. [b] Tox, cytotoxic or antiproliferative
activity; IC50, half-maximal inhibitory concentration of cell viability. Values in
parentheses indicate the percentage of cell viability compared with the con-
trol at the indicated concentration. Data presented correspond to one repre-
sentative experiment. A repetition of the experiment demonstrated a similar
trend of compound activities (see the Supporting Information, Table S1).
2-styrylchromone 3k and chromone 3d, with C5 values of 2.9
to 4.7 μ
tion of 100 μ
M
and only minor effects on cell growth at a concentra-
. Potent induction of the Keap1-Nrf2 pathway
M
at low and possibly physiologically achievable concentrations
makes compounds 3p, 3r, 3o, 3k and 3d promising agents for
further studies as chemopreventive activities.
SAR analyses revealed that both the number and position of
methoxy substituents in the tested benzopyran-4-ones signifi-
cantly influences their Nrf2 induction capacity, as represented
by the C5 value. In the chromone group 3a–d, compounds 3a
and 3d, without methoxy group(s) in ring A, showed more po-
tent Nrf2 induction potential than substituted chromones. We
observed a similar SAR for flavones 3e–g and 2-styrylchromo-
nes 3k–m: addition of methoxy group(s) to the aromatic rings
reduced the capacity to induce the Nrf2 pathway when com-
pared with 3e and 3k lacking methoxy substituents. Finally, in
the most active group of chromanones 3o–r, both the degree
and the position of methoxy substitution influenced inducing
potential. 5-Methoxy-substitution on ring A increased the po-
tency of Nrf2 activators 3p and 3r when compared with the
activity of compounds 3o and 3q with a methoxy substitution
at position C-7 (ring A). The most active compound 3r repre-
sented the highest level of methoxy substitutions, with four
groups placed on two aromatic rings at positions C-5, C-7, C-
3′′′′ and C-4′′′′ (Table 3).
Figure 6. Impact of 3r (top), 3k (middle) and curcumin (bottom) on human
K562 leukemia cell viability and proliferation. DMSO was used as a vehicle
control (VEH); ** p < 0.01, *** p < 0.001.
Among the benzopyran-4-ones, 2-styrylchromone 3k was
the most active, followed by chromanone 3r; none of the other
compounds including 3a, 3e–g and 3l affected either cell pro-
liferation or induced death. Compound 3r presents functional
similarities to curcumin; hence, the CINA pharmacophore could
be a useful tool to further investigate cytotoxic and antipro-
liferative activities.
Antileukemic Activity of Benzopyran-4-ones 3a, 3e–g, 3k–l
and 3r
Chromone 3a, flavones 3e–g, 2-styrylchromones 3k, 3l and
chromanone 3r were then selected for analysis of their impact
on viability and proliferation of human K562 leukemia cells (Fig-
Most significantly, chromanone 3r and 2-styrylchromone 3k
ure 6, Table 4). Chromanone 3r strongly reduced cell viability did not affect the viability of peripheral blood mononuclear
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Table 4. IC50 values of selected benzopyran-4-ones for antileukemic activity
vs. K562 cells.[a]
proliferation of the leukemic cell line K562, whereas PBMCs
from healthy donors were not affected. The spectrum of activi-
ties revealed by 3r and 3k in vitro and their selective toxicity
toward cancer cells supports further investigations of both their
chemopreventive and therapeutic potential. With our synthetic
approach in hand, these promising bioactivities also allow the
design of additional structurally related benzopyran-4-ones, as
minor modifications in substituents appear to affect the anti-
cancer potential.
Compound
Tox. IC50 [μ
M]
24 h
48 h
72 h
3a
> 100
3e
> 100
3f
> 100
3g
> 100
3k
3l
8.6 4.1
4.0 1.2
> 100
4.5 1.9
3r
13.1 4.5
28.3 7.2
7.5 1.0
14.3 3.4
7.9 2.6
15.0 1.0
curcumin
Experimental Section
[a] Tox, cytotoxic or antiproliferative activity; IC50: half-maximal inhibitory
concentration of cell viability.
1,3-Dicarbonyl compounds 2a–r were prepared by using the Baker–
Venkataraman method.[22]
cells (PBMC) from healthy donors (IC50 > 50 μM) (Figure 7). Both
General Procedure for Michael Addition of 1,3-Dicarbonyl Com-
pounds 2a–r on Chromone-3-carboxylic Acid 1: Synthesis of 3-
(HOPO-1)chromones, -flavones, -2-styrylchromones 3a–g, 3j–n
and 3o′, and 2,3-Disubstituted Chromanones 3o–r: Chromone-
3-carboxylic acid 1 (5.26 mmol, 1 g), was added to 1,3-dicarbonyl
compounds 2a–r (5.26 mmol) and a catalytic amount of 4-PPy
(0.26 mmol, 0.04 g, 0.05 equiv.), the reaction mixture was brought
to reflux in chloroform (10 mL). During the required reaction time
(see Table 2), gradual consumption of the starting materials 1 and
2a–r was monitored by TLC (CH2Cl2 or CH2Cl2/light petroleum),
which showed, in most of the cases, several spots, including the
desired product accompanied by the formation of by-products 3a
and 3aa as verified by authentic samples and remaining starting
materials (especially, 1,3-dicarbonyl compounds 2a–r, observations
are made in Table 2). The required reaction time was determined
as the point at which no further change was observed according
to TLC analysis. The reaction was then stopped and the solvent was
removed by evaporation to give a dark-red resinous solid, which
was either purified by silica gel column chromatography and then
recrystallised or recrystallised directly from an appropriate solvent
to afford compounds 3a–g, 3j–n, 3o′ and 3a–r (indications are
mentioned with each compound analytical data). The by-products
3a (dimeric-product) and 3aa (chromone) (when formed, see
Table 2) were only compared to authentic samples by TLC but never
recovered, except in the case of 3h and 3i reactions, for which the
dimeric-compound 3a was produced as the major product.
curcumin and chromanone 3r become cytotoxic in a compara-
ble range of concentrations. These data confirm the selective
bioactivity of 3r and 3k against cancer cells and underline the
potential of both compounds as future lead structures.
Figure 7. Impact of 3k, 3r and curcumin on viability of peripheral blood
mononuclear cells (PBMC) from health donors treated for 24 h. DMSO was
used as a vehicle control (VEH).
Conclusions
Ferric Ion Reducing Ability Assessment: The antiradical ability of
3a–r was measured by assessing their ferric reducing power against
ferric complexes. The FRAP assay[23a] depends upon the reduction
of a ferric tripyridyltriazine (Fe3+–TPTZ) complex to the ferrous tripy-
ridyltriazine (Fe2+–TPTZ) by a reductant at low pH. Trolox was used
Novel benzopyran-4-one-based compounds (chromone, fla-
vone, 2-styrylchromone and chromanone derivatives) have
been prepared by a Baker–Venkataraman synthesis of 1,3-di-
carbonyl compounds followed by a Michael addition tandem
reaction of these compounds on chromone-3-carboxylic acid
under organobase catalysis. The designed benzopyran-4-ones
sharing Michael acceptor functionalities were screened for radi-
cal scavenging potential and cytoprotective properties by in-
ducing the Nrf2 pathway, and antileukemic activity. These bio-
logical effects were most potently achieved by two classes of
compounds, chromanones 3o–r, sharing the curcuminoid bio-
activator CINA moiety, and 2-styrylchromones 3k–m, sharing
the HOPO bioactivator function. The best radical scavengers
were chromanones 3o–r, with a very high activity, probably due
to their CINA moiety. Moreover, chromanones 3p and 3r and 2-
styrylchromones 3k potently activated the Nrf2 response in the
as a reference. Compounds were tested at 25 μ
reagent was prepared as required by mixing acetate buffer (300 m
25 mL, pH 3.6), TPTZ solution (10 m in HCl, 2.5 mL, 40 m ), and
FeCl3·6H2O (20 m , 2.5 mL). Freshly prepared FRAP reagent was
M. Working FRAP
M,
M
M
M
warmed at 37 °C for 30 min and added to the samples. Absorbance
of (Fe2+–TPTZ) was measured at 593 nm after 30 min. Each sample
was tested two times with three measures in each experiment.
DPPH Scavenging Activity Measurement: The antiradical activity
˙
of 3a–r was measured by assessing their DPPH (2,2-diphenyl-1-pi-
˙
crylhydrazyl) free radical-scavenging activity. DPPH is a stable radi-
cal displaying an intense violet colour with a maximum absorbance
˙
at 515 nm. In the presence of an antioxidant compound, DPPH is
neutralised to a compound with slight yellow colour, which causes
an absorbance decrease.[23b] This modification is proportional to
the antioxidant capacity of the compound and is measured by spec-
AREc32 cell line at low micromolar concentrations (C5 <3 μ
M),
which might be physiologically achievable. At slightly higher
concentrations, compounds 3r and 3k reduced viability and trophotometry. The degree of discoloration induced by a tested
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sample is related to that induced by Trolox, allowing its antioxidant Aldrich), aliquoted and stored at –20 °C upon treatment. Cells in
˙
potent to be expressed as Trolox equivalents. DPPH was used at a
final concentration of 120 μ in 1 mL. Compounds were tested at
10 μ
from 5 to 70 μ
exponential growth phase were treated at a concentration of
200,000 cells/mL. Control cells were treated with the same volume
M
M
. The range of Trolox concentrations for calibration spread of DMSO as the one required for compounds. Cell proliferation and
M
. The optical density at 515 nm was read after
viability as well as cell cycle analyses were measured as previously
incubation in the dark for 30 min. The absorbance of pure com-
pounds suitably diluted was subtracted. Each sample was tested
two times in triplicate.
reported.[24]
Acknowledgments
AREc32 (Human Mammary Epithelial Adenocarcinoma MCF-7)
Cells: AREc32 (kindly provided by Prof. Roland Wolf, University of
Dundee) is a reporter cell line derived from the human mammary
epithelial adenocarcinoma MCF-7 cells. It responds strongly and se-
lectively to Nrf2, which, upon activation, binds to the ARE sequence
present in eight copies in the promoter region of luciferase gene.
This drives luciferase expression and can be assessed by measuring
enzyme activity.[16] AREc32 cells were cultured at 37 °C and 5 %
CO2 in a humidified atmosphere in complete DMEM medium sup-
The authors thank the Fundação para a Ciência e a Tecnologia
(FCT), Portugal (grant numbers UID/QUI/00062/2013, UID/CTM/
50011/2013) the European Union (EU), the Quadro de Referên-
cia Estratégico Nacional (QREN), the Fundo Europeu de Desen-
volvimento Regional (FEDER) and the Programa Operacional
Factores de Competitividade (COMPETE) for funding the Or-
ganic Chemistry Research Unit, the Associate Laboratory Centro
de Investigação em Materiais Cerâmicos e Compósitos – CICECO
and the Portuguese National NMR Network (RNRMN). European
Community's Seventh Framework Programme (FP7/2007-20139,
grant agreement number 215009) is also acknowledged for fi-
nancial support. The General Directorate for Scientific Research
and Technological Development-DGRSDT of Algeria is thanked
for financial support. The authors further wish to thank FCT for
specific funding toward the purchase of the single-crystal X-ray
diffractometer. O. T. appreciates support for his post-doctoral
position through the project New Strategies Applied to Neuro-
pathological Disorders (CENTRO-07-ST24-FEDER-002034), co-
funded by QREN, Mais Centro-Programa Operacional Regional
do Centro and the EU, FEDER. B. O. thanks RSL, Luxembourg,
for her post-doctoral grant. Thanks are also due to the Fonda-
tion de Recherche Cancer et Sang, the Recherches Scientifiques
Luxembourg association, the Een Haerz fur Kriibskrank Kanner
association, the Action Lions Vaincre le Cancer association, In-
terreg IVA project Corena, and the Te´le´vie Luxembourg for
funding LBMCC. M. D. and B. O. also thank the Research Insti-
tute of Pharmaceutical Sciences by the NRF of the MEST of
Korea for the Tumor Microenvironment GCRC 2012-0001184
grant and by the Brain Korea (BK21) PLUS program for funding.
The AREc32 cell line used in this study was kindly provided by
Professor Roland Wolf (University of Dundee, UK) and the Can-
cer Research UK.
plemented with 10 % heat-inactivated FCS, 2 m
M glutamine,
100 U mL–1 penicillin and 100 μg mL–1 streptomycin (medium and
all supplements from Gibco, US), and maintained under antibiotic
selection (0.8 mg mL–1 G418; Sigma, Germany).
Luciferase Reporter Assay on AREc32 Cells Stimulated with Ben-
zopyran-4-one-Based Polyphenolics 3a–r: For the detection of
Nrf2 induction, the luciferase activity in AREc32 cells was measured.
AREc32 cells were seeded at a density of 9 × 103 per well in 50 μL
growth medium in 384-well plates. After 26 h, compounds were
added to obtain final concentrations in the range between 1.56–
100 μM. Each concentration was tested in quadruplicate. An equal
volume of vehicle was added to the control wells (0.016–1 %
DMSO). The reporter assay was performed as described previ-
ously.[16] For normalisation, all values of luciferase activity were di-
vided by vehicle control values, yielding the fold change relative to
the control. Furthermore, the luciferase activity was normalised to
biomass content. Concentration leading to increase of luciferase ac-
tivity by fivefold (C5) was determined by fitting a logistic dose re-
sponse curve to the experimental data (Table Curve v1.0, Jandel
Scientific).
Cytotoxicity Assay on AREc32 Cells Stimulated with Benzo-
pyran-4-one-Based Polyphenolics 3a–r: Biomass content was as-
sessed by sulforhodamine B (SRB, Sigma, Germany) staining.[16b]
Briefly, AREc32 cells were seeded and treated as for the luciferase
reporter assay. Detached cells in the supernatant were discarded
and attached cells were fixed with 10 % trichloroacetic acid for
30 min at 4 °C. The plate was then rinsed with distilled water, dried
and stained for 15 min with a 0.4 % solution of SRB in 1 % acetic
acid, washed several times with 1 % acetic acid and dried again.
Keywords: Medicinal chemistry · Drug discovery · Anticancer
agents · Oxygen heterocycles · Domino reactions · Michael
addition · Antioxidants
The dye was dissolved with 10 mM TRIS base followed by the meas-
urement of OD at 490 and 515 nm (SpectraMax M5e Microplate
reader, Molecular Devices, US). The concentrations of compounds
giving cell staining below 50 % of control cells were considered as
cytotoxic. The concentration leading to a decrease of cell numbers
to 50 % (IC50) relative to solvent treated cells was determined by
fitting a logistic dose response curve to the experimental data
(Table Curve v1.0, Jandel Scientific).
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Published Online: January 15, 2016
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim