Synthesis and SAR Study of Anticancer Protoflavone Derivatives: Investigation of Cytotoxicity and Interaction with ABCB1 and ABCG2 Multidrug Efflux Transporters

Article abstract of DOI:10.1002/cmdc.201700225

There is a constant need for new therapies against multidrug-resistant (MDR) cancer. Natural compounds are a promising source of novel anticancer agents. We recently showed that protoflavones display activity in MDR cancer cell lines that overexpress the P-glycoprotein (P-gp) drug efflux pump. In this study, 52 protoflavones, including 22 new derivatives, were synthesized and tested against a panel of drug-sensitive parental cells and their MDR derivatives obtained by transfection with the human ABCB1 or ABCG2 genes, or by adaptation to chemotherapeutics. With the exception of protoapigenone, identified as a weak ABCG2 substrate, all protoflavones bypass resistance conferred by these two transporters. The majority of the compounds were found to exhibit mild to strong (up to 13-fold) selectivity against the MCF-7_Dox and KB-V1 cell lines, but not to transfected MDR cells engineered to overexpress the MDR transporters. Our results suggest that protoflavones can overcome MDR cancer by evading P-gp-mediated efflux.

Full text of DOI:10.1002/cmdc.201700225

Accepted Article
Title: Synthesis and SAR Study of Novel Anticancer Protoflavone
Derivatives - Investigation of Cytotoxicity and Interaction with the
ABCB1 and ABCG2 Multidrug Efflux Transporters
Authors: Balázs Dankó, Szilárd Tóth, Ana Martins, Vágvölgyi Máté,
Norbert Kúsz, Joseph Molnár, Fang-Rong Chang, Yang-
Chang Wu, Gergely Szakács, and Attila Hunyadi
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201700225
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ChemMedChem
Synthesis and SAR Study of Novel Anticancer Protoflavone Derivatives –
Investigation of Cytotoxicity and Interaction with the ABCB1 and ABCG2
Multidrug Efflux Transporters
Balázs Dankó,[a],# Szilárd Tóth,[b],# Ana Martins,[c],† Máté Vágvölgyi,[a] Norbert Kúsz,[a] Joseph
Molnár,[c] Fang-Rong Chang,[d],[e],[f] Yang-Chang Wu,[g],[h],[i],[j] Gergely Szakács,[b],[k] and Attila
Hunyadi[a],[l],
*
[a] Institute of Pharmacognosy, University of Szeged, Szeged, Hungary;
[b]
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences,
Budapest, Hungary;
[c]
Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of
Szeged, Szeged, Hungary;
[d] Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan, R.O.C.;
[e] Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, R.O.C.;
[f] R&D Center of Chinese Herbal Medicines & New Drugs, College of Pharmacy, Kaohsiung Medical
University, Kaohsiung, Taiwan, R.O.C.;
[g] School of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan, R.O.C.;
[h] Research Center for Chinese Herbal Medicine, China Medical University, Taichung, Taiwan R.O.C.;
[i] Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung,
Taiwan, R.O.C.
[j] Center of Molecular Medicine, China Medical University Hospital, Taichung, Taiwan, R.O.C.;
[k] Institute of Cancer Research, Medical University Vienna, Vienna, Austria
[l] Interdisciplinary Centre for Natural Products, University of Szeged, Szeged, Hungary
# Equal contributions for the first two authors
† Current address: Synthetic Systems Biology Unit, Institute of Biochemistry, Biological Research
Centre, Temesvári krt. 62, 6726 Szeged, Hungary.
* Correspondence to: Attila Hunyadi, Tel: +3662546456, Fax: +3662545704, email:
hunyadi.a@pharm.u-szeged.hu
1
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ChemMedChem
Abstract
There is a constant need for new therapies against multidrug resistant (MDR) cancer. Natural
compounds represent a promising class of novel anticancer agents. Recently, we have shown
that protoflavones display activity in multidrug resistant cancer cell lines overexpressing the
drug efflux pump P-glycoprotein. In the present study, 52 protoflavones, including 22 new
derivatives were synthesized and tested against a panel of sensitive parental cells and their MDR
derivatives obtained by transfection with the human ABCB1 or ABCG2 genes, or by adaptation
to chemotherapeutics. With the exception of protoapigenone, identified as a weak ABCG2
substrate, all protoflavones bypass resistance conferred by these two transporters. The majority
of the compounds exhibited mild to strong (up to 13 fold) selectivity against the MCF-7Dox and
KB-V1 cell lines, but not to transfected MDR cells engineered to overexpress the MDR
transporters. Our results suggest that protoflavones can overcome cancer multidrug resistance
by evading efflux by P-glycoprotein.
Keywords: protoflavone, MDR cancer, collateral sensitivity / cross-resistance, ABCB1 / P-
glycoprotein, ABCG2 / BCRP
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Introduction
Cancer is among the leading causes of morbidity and mortality worldwide. According to the
World Cancer Report, 8.2 million cancer-related deaths were registered in 2012, and it is
expected that annual cancer cases will rise from 14 million in 2012 to around 22 million within
the next two decades.[1] Resistance to chemotherapy and molecularly targeted therapies is a
major problem facing current cancer research.[2] Despite the availability of a broad range of
diverse anticancer compounds with new mechanisms and molecular targets, cancer is often
incurable due to the development of drug resistance.[3] Resistance can rapidly develop even in
cases when the tumor initially responds to chemotherapy. Multi-drug resistance (MDR) can
emerge as a result of reduced uptake or increased efflux of cytostatic agents – the latter is
mediated by ATP-binding-cassette (ABC) proteins, primarily by P-glycoprotein (P-gp;
ABCB1) and ABCG2, which confer resistance to a wide variety of compounds.[4],[5] There is a
constant need for novel chemotherapeutics with marked and selective antitumor activity that
can overcome resistance mediated by these transporters. The unfavorable prognostic impact of
P-glycoprotein expression in several cancers has prompted overwhelming research efforts
aimed at the clinical development of high affinity efflux inhibitors that were shown to overcome
MDR in in vitro models. Unfortunately, even after decades of intensive research, a clinically
effective inhibitor has not been identified. Recently, the discussion has shifted to alternative
strategies, either to bypass the transporters or to exploit the collateral sensitivity (CS) of MDR
cells.[6] Recent discoveries have shown that it is possible to invert the selective advantage of
resistant cells to reverse the evolution of resistance.[7] For example, MDR-selective compounds
were shown to specifically target ABC transporter over-expressing MDR cancer cells by
exploiting the Achilles’ heel conferred by the overexpression of the transporters.[8],[9],[10]
Our review of the literature identified several natural compounds that were reported to elicit
preferential toxicity against MDR cells.[6] For example, the 4´-hydroxyflavone apigenin was
identified in a screen as a specific killer of drug-selected H69AR cells and MRP1-transfected
HeLa cells.[11] Flavonoids are naturally derived compounds that display both anti- and
prooxidant properties. Flavonoids have been used in cancer chemoprevention and
chemotherapy. A particularly interesting, rare group of natural flavonoids with a high antitumor
potential contains protoflavones. Typically derived from ferns, protoflavones contain a non-
aromatic, usually p-quinol B-ring or its di- or tetrahydro derivative. Based on the most
frequently occurring chemical nomenclature, herein we refer to the flavone skeleton containing
a 1´-OH group and a 2´,5´-dien-4´-one moiety in its B-ring as the “protoflavone” skeleton.
Protoflavones can formally be derived from 4´-hydroxyflavones, like apigenin (1), and some,
e.g. protoapigenone (2), the protoflavone analog of 1, have been described as potent anticancer
agents in vitro and in vivo. We have recently reviewed the chemistry and bioactivity of
protoflavones.[12] The proapoptotic activity of protoflavones is mediated by oxidative stress[13]
and the inhibition of ATR-dependent signaling.[14] We have previously shown that 6-
methylated protoflavone derivatives exert mild selective cytotoxicity against a murine
lymphoma cell line transfected with the human ABCB1 transporter, while other protoflavones,
derived from apigenin, genkwanin or β-naphthoflavone, did not exhibit such a selectivity.[15]
Furthermore, protoapigenone and its 1´-O-butyl- and propargylether, the β-naphthoflavone
analog WYC0209, 6-methylprotoflavone and 6-bromoprotoflavone showed selective
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ChemMedChem
cytotoxicity against certain MDR cancer cell lines, such as NCI-H460 human non-small cell
lung carcinoma cells adapted to doxorubicin, U87 human glioma and DLD1 human colorectal
cells, both adapted to paclitaxel.[16] On the other hand, cross-resistance (CR) to protoflavones
was observed in C6 rat glioma cells adapted to carmustine, and CS/CR pattern appeared to be
in line with altered antioxidative capacity of the MDR cells as compared to their parental cell
lines.[16]
Based on these results our aim was to systematically explore the cytotoxicity and antitumor
potential of further protoflavone derivatives. In particular, we characterized the anticancer
activity of a total of 52 compounds in a diverse panel of cancer cell lines including MDR
derivatives expressing ABCB1 or ABCG2.
Results
Thirty-seven protoflavones and protoflavone 1´-O-alkyl ethers were synthesized from apigenin
(1), genkwanin, 4´-hydroxy-6-methylflavone, 4´-hydroxy-6-methoxyflavone and 4´-hydroxy-
β-naphthoflavone, based on the synthetic route we have previously published for compounds
2-9,[17] 11-24[15] and 31-38.[17] Briefly, an oxidative de-aromatization was performed by a
common hypervalent iodine reagent, [bis(trifluoroacetoxy)iodo]benzene (PIFA) in acetonitrile
in the presence of water or the alcohol to be coupled at position C-1´. Among these compounds,
protoapigenone 1´-O-benzylether (10) and the 6-methoxylated derivatives (25-30) were
obtained as new protoflavones; synthesis and structures of the compounds are presented in
Scheme 1.
Scheme 1. Synthesis of protoflavones from commercially available 4´-hydroxyflavonesa
R1=OH, R2=H, R3=OH
2: R=H
3: R=Me
4: R=Et
7: R=But
8: R=Allyl
9: R=Propargyl
i.
5: R=Pro 10: R=Bn
6: R=i-Pro
Apigenin (1)
R1=OH, R2=H, R3=OH
Genkwanin
R1=OMe, R2=H, R3=OH
R1=OMe, R2=H, R3=OH R1=H, R2=Me, R3=H R1=H, R2=OMe, R3=H
11: R=H
18: R=H
25: R=H
12: R=Me
13: R=Et
19: R=Me
20: R=Et
26: R=Me
27: R=Et
4 -Hydroxy-6-methylflavone
R1=H, R2=Me, R3=H
14: R=Pro
15: R=But
16: R=Allyl
17: R=Propargyl
21: R=Pro
22: R=But
23: R=Allyl
24: R=Propargyl
28: R=But
29: R=Allyl
30: R=Propargyl
4 -Hydroxy-6-methoxylflavone
R1=H, R2=OMe, R3=H
31: R=H
32: R=Me
33: R=Et
i.
34: R=Pro
35: R=i-Pro
36: R=But
37: R=Allyl
38: R=Propargyl
4 -hydroxy-β-naphthoflavone
a Reagents: (i.) CH3CN/ROH 9/1, PIFA (2 eq).
Total synthesis of a set of various 6-substituted protoflavones was achieved in 4-6 steps. In
order to obtain starting materials (i.e. 5´-ethyl-2´-hydroxyacetophenone, 41; and 5´-pentyl-2´-
hydroxyacetophenone; 42) for our 6-ethyl and 6-pentyl substituted target compounds, the
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10.1002/cmdc.201700225
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appropriate p-substituted phenols were acetylated and subjected to Fries-rearrangement
reaction under the condition of dry AlCl3 in dichloromethane.[18] The resulting 2´-
hydroxyacetophenones and those commercially available with a 5´-ethoxy or -bromo
substituent (43 and 44, respectively) were utilized in Claisen-Schmidt condensation reactions
with p-benzyloxybenzaldehyde to yield chalchones (45-48), which, after performing ring
closure with iodine in DMSO, yielded the corresponding 6-substituted 4´-benzyloxyflavones
(49-52). The 6-bromo substituted compound (52) was subjected to Suzuki coupling in order to
obtain the corresponding 6-phenylflavone (53). Debenzylation of the flavonoids obtained this
way and subsequent oxidative de-aromatization of the flavones 54-58 with PIFA, as described
above, allowed us to obtain the protoflavones with various substituents at positions C-6 and C-
1´ (59-73). Scheme 2 summarizes the total synthetic procedure.
Scheme 2. Total synthesis of 6-substituted protoflavone derivativesa
iii.
iv.
49: R1=Et
45: R1=Et
50: R1=Pent
51: R1=OEt
52: R1=Br
46: R1=Pent
47: R1=OEt
48: R1=Br
ii.
41: R1=Et
v.
42: R1=Pent
43: R1=OEt
44: R1=Br
53: R=Ph
39: R1=Et
vi.
40: R1=Pent
i.
vii.
R1=OEt
R1=Ph
59: R=H
68: R=H
R1=Et or Pent
60: R=Me
61: R=Et
62: R=Pro
63: R=i-Pro
64: R=But
69: R=Me
70: R=Et
71: R=Pro
72: R=i-Pro
73: R=But
54: R1=Et
55: R1=Pent
56: R1=OEt
57: R1=Br
58: R1=Ph
65: R1=Et, R=H
66: R1=Pent, R=H
67: R1=Br, R=H
a
Reagents: (i.) (CH3CO)2O, cc H2SO4; (ii.) AlCl3; (iii.) EtOH, 4-benzyloxybenzaldehyde, 50% KOH/H2O; (iv.)
I2, DMSO; (v.) Phenylboronic acid, K2CO3, Tetrakis(triphenylphosphine)palladium(0); (vi.) 10% Pd-C/H2; (vii.)
CH3CN/ROH 9/1, PIFA (2 eq).
In the first set of experiments, the cytotoxicity of compounds 2-38 and 59-73 were tested in two
MDR/sensitive cancer cell line pairs (parental L5178 mouse lymphoma cells and L5178B1 cells
engineered to overexpress the human ABCB1 protein; parental MCF-7 breast cancer cells and
the doxorubicin resistant derivative MCF-7Dox, overexpressing P-gp). The fraction of IC50
values obtained in P-gp negative vs. positive cells served as a quantification of the MDR
selective effect (selectivity ratio, SR). Accordingly, SR ≤ 0.5 indicated that the compound is
subject to P-gp-mediated resistance, whereas SR ≥ 2 suggested that the P-gp expressing cells
demonstrate collateral sensitivity against the tested protoflavone derivative. Results of the
cytotoxicity testing on the L5178 and MCF-7 models are summarized in Figure 1; detailed data
are presented in Supplementary Table S1.
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10.1002/cmdc.201700225
ChemMedChem
A
B
S R = 2
6 . 5
6 . 0
5 . 5
5 . 0
4 . 5
4 . 0
7 . 0
S R = 2
S R = 0 . 5
6 . 5
6 . 0
5 . 5
5 . 0
4 . 5
S R = 0 . 5
D
o x
D
o x
4 . 0
4 . 5
5 . 0
M C F - 7
0
5 . 5
6 . 0
4 . 5
5 . 0
5 . 5
6 . 0
6 . 5
7 . 0
p I C
p I C
L 5 1 7 8
0
5
5
Figure 1. Cytotoxicity of protoflavones 2-38 and 59-73. pIC50 values were derived from dose
response curves obtained from cell viability experiments on L5178/L5178B1 mouse lymphoma
non-
cells (A) and MCF-7/MCF-7Dox cells (B). SR=Selectivity Ratio, calculated as IC50
MDR/IC50MDR; n=3-4; Dox: doxorubicin.
The above results indicate that the synthesized protoflavone derivatives possess significant
toxicity in the two cell line pairs. Interestingly, MCF7 cells were in general more resistant.
Whereas the expression of ABCB1 did not modify the sensitivity of L5178B cells in comparison
with the parental L5178 cell line, MCF-7Dox cells showed collateral sensitivity to several
derivatives, with SR values exceeding 5 in the case of compounds 16, 18-22, 37, 68-71 and 73.
To substantiate the role of ABC transporters in the MDR-selective toxicity of the compounds,
additional MDR models were included in the study. The cytotoxic activity of compounds 2-11,
18, 31-38, 66 and 68-73 were tested in four additional MDR/sensitive cell line pairs, including
A431, A431B1, A431G2, MES-SA, MES-SA/Dx5, KB-3-1 and KB-V1. These compounds
represent a diverse sub-set of derivatives of the naturally occurring protoapigenone (2) and
protogenkwanone (11), analogs of the synthetic WYC0209 (31) identified as a potential lead in
previous studies,[12] as well as 6-methyl- 6-pentyl- and 6-phenyl derivatives (18, 66 and 68-73)
aiming to further explore SAR at C-6. The results are shown in Figures 2-3; detailed data are
available as supplementary information (Tables S2-S3).
The tested protoflavone derivatives were equally toxic to A431, A431G2 or A431B1 cells (IC50
values ranged from 0.60 µM to 7.27 µM), with the exception of protoapigenone (2), suggesting
that the compounds tested herein are able to bypass ABCB1 or ABCG2. Resistance of A431G2
cells to compound 2 was abolished in the presence of tariquidar, confirming that protoapigenone
is an ABCG2 substrate (Figure 2).
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Protoapigenone (2)
Mitoxantrone
B
A
6.5
6.0
5.5
5.0
4.5
A431B1
A431G2
SR=2
12
10
8
*
**
**
SR=0.5
**
ns
6
Mx
6.0
4
2
ns
2
0
5.0
5.5
6.5
)
)
)
)
1
1
3
3
1 G2
1 G2
4
4
TQ
TQ
pIC50 A431
(
(TQ
1 G2
(
(TQ
1 G2
3
3
A
A
4
4
1
1
A
A
3
3
4
4
3
3
A
A
4
4
A
A
Figure 2. A. Cytotoxic activity of selected protoflavones against A431 (parental) cell line and
two MDR derivatives engineered to overexpress ABCB1 (A431B1) or ABCG2 (A431G2).
SR=Selectivity Ratio, calculated as IC50sensitive/IC50MDR; n=3-4; Mx: mitoxantrone. B. IC50
values of 2 (protoapigenone) and mitoxantrone in A431 and A431G2 cell lines in the presence
and absence of 1 µM tariquidar, an ABCG2 efflux inhibitor.
B
A
7.0
6.5
6.0
5.5
5.0
SR=2
6.5
6.0
5.5
5.0
SR=2
SR=0.5
SR=0.5
Vbl
Dox
6.5
5.0
5.5
6.0
5
6
7
8
pIC50 MES-SA
pIC50 KB-3-1
Figure 3. pIC50 values measured in doxorubicin-selected MES-SA/Dx5[19] and vinblastine-
selected KB-V1[20] cells compared to the pIC50 values of MES-SA and KB-3-1 cells (A and B,
respectively). Two compounds were selectively toxic against both MES-SA/Dx5 and KB-V1
cells (2 and 66), and none of the protoflavones showed substrate-like characteristics; Dox:
doxorubicin, Vbl: vinblastine.
Finally, we tested the interaction of the compounds with Pgp to reveal if any of the compounds
inhibit drug efflux. ABCB1 function was characterized using the calcein accumulation assay.[21]
Each derivative was assayed at two concentrations in the presence of the fluorescent indicator.
Except for the 6-phenylprotoflavone series (68-73), which showed moderate inhibition at
20 µM (14-46 %; see supplementary Table 4), none of the compounds inhibited the efflux of
calcein AM by P-gp (Figure 4).
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ChemMedChem
A
B
100
80
60
40
20
0
100
2 M
2 M
20 M
R=H
R1=Ph
20 M
80
60
40
20
0
2
11 18 31 65 66 68
68
69
70
(R=Et)
71
72
73
(R=Bu)
(R=i-Pr)
(R=Me)
(R=Pr)
(R=H)
R:
H
Me
Pr
i-Pr
Bu
Et
.
Figure 4. Relative inhibition of calcein AM efflux by A compounds 2, 11, 18, 31, 65, 66 and
68 (where R=H refers to Scheme 1 and Scheme 2), and B derivatives of compound 68 (6-
phenylprotoflavones). Relative inhibition was calculated from mean calcein intensities as
[100*(sample - negative control)/(positive control - negative control)]. 20 µM verapamil was
used as positive control, corresponding to full inhibition (100%).
Discussion
This work was initiated with the aim to explore relevant structure-activity relationships of
protoflavones with various substituents at the A-ring and particularly at C-6. Following the
preparation of acetophenones 41 and 42, a straightforward total-synthetic strategy[25] was
applied to obtain 6-substituted protoflavones. It is worth mentioning that, even though related
publications typically describe the use of a catalytic amount of iodine for the ring closure to
obtain the flavone skeleton, utilizing a larger, 1 equivalent amount is far more efficient.
Structure elucidation of the protoflavones was straightforward based on the mass and 1H NMR
spectra. The expected change in the molecular mass, and, in case of the 1´-O-alkyl derivatives,
1
the appearance of the characteristic H NMR signals and coupling pattern of the side chain
proved the successful linking of water or alcohol. The build-up of the protoflavone type B-ring
was evidenced by the change in the coupling constant of the two dublets of H-3´/H-5´ and H-
2´/H-6´ from ca. 8.8 Hz to ca. 10.0 Hz, together with the remaining H-3 singlet and practically
unchanged A-ring signals in the 1H NMR spectrum.
According to their B-ring substitution, cytotoxicity of the protoflavones on the utilized cell lines
typically followed the previously observed structure-activity relationship: in most of the cases,
1´-OH substituted compounds were more toxic than those with 1´-alkoxy moieties and the
isopropyl-ethers were the least cytotoxic derivatives. This, however, did not apply for
protogenkwanone and its analogs (11-17): protogenkwanone 1´-O-methylether (12) exerted a
stronger activity on the mouse lymphoma cells than 11. Moreover, an at least two carbons long
side-chain was necessary for this series of compounds to be slightly toxic on MCF-7 cells.
Presence of a non-branching propyl or butyl ether side chain at the C-1´ of protoapigenone (as
in compounds 5 and 7) was previously found to be preferable for a strong cytotoxic activity.[17]
This was also observed in the present study in most cell lines with the exceptions of the L5178
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ChemMedChem
/ L5178B1 and the MCF-7 / MCF-7Dox cell line pairs. Moreover, the introduction of a 1´-
benzyloxy moiety (as in compound 10) to protoapigenone also resulted in an increased toxicity
in the A431 cell line and its MDR sub-cell lines, as well as in the KB-3-1 and KB-V1 cells.
This provides further evidence for the importance of the size and/or lipophilicity of the
substituent at C-1´ and suggests that a larger branching and/or unsaturated alkyl side-chain
might also lead to an increased cytotoxicity, despite the generally lower activity of the 1´-O-
isopropyl substituted derivatives as compared to those with linear alkyl chains.
Our attempt to increase the mild selective toxicity of 6-methylprotoflavones (18-24) previously
observed in ABCB1 transfected L5178 cells[15] by introducing various C-6 substituents revealed
one single compound reaching our chosen threshold of relevance against the L5178B1 cells, 6-
methoxyprotoflavone 1´-O-allylether (29). On the other hand, while all 6-methoxy compounds
also showed tendency for such selectivity, other new derivatives showed decreased selective
cytotoxicity against this cell line (Table S1). Similarly, none of the tested compounds, including
6-methylprotoflavone (18), showed selective toxicity against ABCB1 transfected cell lines,
including A431B1 (Table S2) and MDCK-IIB1 (not shown). Despite the equal toxicity of the
studied compounds on parental and Pgp-transfected cell lines, several derivatives proved
selectively toxic against MDR cell lines overexpressing P-gp as a result of long-term drug
selection. In particular, MCF-7Dox cells (adapted to doxorubicin) showed collateral sensitivity
to most compounds except for 4, 31, 35 and 59-65, with structural differences of the A-ring
clearly influencing activity (Table S1). Compound 68, 6-phenylprotoflavone, for example,
showed a remarkable, 13.2 fold selective cytotoxicity, while its β-naphthoflavone analog (31),
where a fused aromatic ring is connected to the A-ring at the C-5/C-6 position, was non-
selective. By comparing the selectivity ratios of compounds with different C-6 substituents, a
clear SAR of the following order was observed: Ph > Me > OMe ≈ Pentyl, while the ethoxy
substituted protoflavones (59-64) and the ethyl substituted compound 65 were non-selective.
No such general SAR could be concluded for the C-1´ substituents, except for the lower
selectivity observed for the isopropyl ether derivatives 35 and 72. Interestingly, in case of the
MES-SA / MES-SA/Dx5 cell line pair where the MDR sub-cell line was also obtained by
adaptation to doxorubicin[19], collateral sensitivity was observed only for the classical, 1´-OH
containing protoflavones (2, 11 18 and 66, but SR was below threshold for 31 and 68) and not
for any of the 1´-O-alkylprotoflavones. Furthermore, the KB-V1 cell line, obtained from KB-
3-1 by adaptation to vinblastine[20], also presented marginal CS towards most of the
protoflavones, although SR values for several compounds fell just below the 2-fold threshold
(Figure 3B, Table S3).
Statistical significance of the SAR was tested from two angles. Compounds were grouped either
according to their A-rings or their substituents at C-1´, and the SR values of these groups were
compared by one-way ANOVA* followed by Bonferroni’s post hoc test. No differences were
observed for the C-1´ substituents on any of the cell lines, not even when the data were
normalized to the average of their corresponding series (i.e. analogs with the same A-ring).
* Each group containing at least 7 data points passed the Shapiro-Wilk normality test, suggesting the
normal distribution of SR values under the influence of the presented chemical variations. Therefore,
ANOVA is suitable for the statistical evaluation of these datasets.
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However, the different A-ring containing protoflavone derivatives showed significant
differences in their SR values on the MCF-7 / MCF-7Dox cell line pair; results are presented in
Figure 5.
15
d
bd
10
5
ac
ab
ac
ac
c
2.0
0.5
0
t
e
n
Ge
F
h
P
N
PA
M
Me
-
OE
6
B
O
6
6
Figure 5. Selectivity ratio (SR) values for protoflavone analogs containing the same A-rings
on the MCF-7 / MCF-7Dox cell line pair. Box-and-whisker plots represent medians, first and
third quartiles and ranges; SR≥2.0 and SR≤0.5 represent CS and CR, respectively; different
lower case letters represent datasets with statistically significant differences (i.e. groups with
overlap in their marking are not significantly different) at p≤0.05 by one-way ANOVA
followed by Bonferroni’s post-hoc test.
All tested compounds were found similarly cytotoxic on the L5178 and the L5178B1 cell lines,
with a strong correlation between the two (Spearman r=0.9544). Interestingly, IC50 values on
the MCF-7Dox cell line also showed a good correlation to those on the parental mouse lymphoma
cell line (Spearman r=0.7691), while the same correlation for MCF-7 was much weaker with
several outliers (Spearman r=0.5812; without outliers, i.e. compounds 8, 11-17, 19-24, 37, 68-
71 and 73: r=0.7420). For a graphical interpretation of these correlations, see supplementary
Figure S1.
These results suggest that the SR associated with the compounds on the MCF-7/MCF-7Dox is
more a result of the resistance of MCF-7 to protoflavones, than the sensitivity of MCF-7Dox
.
MCF-7 cells appear to be particularly resistant to the 7-methoxy group containing
protogenkwanone derivatives (11-17), 6-methylprotoflavones (19-24), 6-phenylprotoflavones
(68-71 and 73) and 1′-allyl group containing analogs (8 and 37), in line with the results
presented in Figure S1. As such, adaptation of MCF-7 cells to doxorubicin has apparently
resulted in the loss of initial resistance to protoflavones as an evolutionary cost of acquiring the
MDR phenotype, and this manifested as collateral sensitivity.
Collateral sensitivity is causally linked to the adaption of MDR cells to a chemotherapeutic and
may involve metabolic modifications, the upregulation of receptors[22] or the modulation of the
redox homeostasis[23]. One limitation of studies relying on MDR cell lines is that the
contribution of MDR pumps, versus other acquired cellular alterations, cannot be
10
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10.1002/cmdc.201700225
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delineated.[8],[9],[24] Collateral sensitivity of the MDR cell lines analyzed in this study indicates
that resistance to doxorubicin or vinblastine may result in cellular alterations that render the
cells susceptible to the protoflavone derivatives. However, in contrast to MDR-selective
compounds,[10] protoflavone derivatives do not selectively target cells engineered to
overexpress P-glycoprotein, suggesting that the increased toxicity observed in the MDR cells
is not conferred by the efflux pumps. This was also supported by our observation that selectivity
ratios did not decrease significantly when compounds 2, 11, and 18 were tested on the MES-
SA / MES-SA/Dx5 cell line pair in the presence of tariquidar (data not presented).
It is important to point out that the lack of cross-resistance to most protoflavones in all MDR
cell lines studied here indicates that these compounds can overcome MDR through bypassing
efflux that is mediated by ABCB1 or ABCG2. SAR of the cytotoxic activity concerning the 6-
substituents appears to differ from cell line to cell line, for example an order of Me > Et ≈ Pent
≈ Ph > Br > OEt > OMe can be recognized on L5178 and L5178B1, Pent > Ph ≈ Me on KB-3-1
and KB-V1, while similar activities are exerted by 6-pentyl (66), 6-phenyl (68) and 6-methyl
(18) compounds on MES-SA and MES-SA/Dx5. From a general overview, 6-alkyl substituted
protoflavones appear to be somewhat more favorable anticancer agents over 6-alkoxy ones,
even though nearly all compounds presented here can be considered as valuable leads against
resistant cancers. As an interesting exception to this, however, resistance to protoapigenone (2)
was observed in the ABCG2 transfected A431G2 cell line, and a tendency for marginal
resistance appeared also to its 1′-O-alkyl ethers. Resistance of this cell line to protoapigenone
(2) markedly decreased in the presence of tariquidar, strongly suggesting that compound 2 is
an ABCG2 substrate. ABCG2 did not confer resistance towards any of the other compounds
including protogenkwanone (11), which differs from 2 only in its 7-methoxy group. This
suggests that a non-substituted phenolic OH group at C-7 is necessary for protoflavones to be
recognized by this transporter.
Conclusions
Our in vitro studies on various A-ring and 1′-substituted protoflavones revealed 6-
methoxyprotoflavone 1′-O-allyl ether (29) as an antitumor agent with a mild MDR selectivity
(SR=2.0) in a murine lymphoma cell line transfected with the human ABCB1 efflux transporter.
The ability of protoflavones to evade efflux-mediated MDR was confirmed both in ABCB1 and
ABCG2 expressing cell lines, with the exceptions of protoapigenone (2) which was identified
as an ABCG2 substrate. MDR selective cytotoxicity was observed for most of the tested
protoflavones in a breast cancer cell line adapted to doxorubicin (MCF-7Dox) and SAR revealed
importance of the A-ring substitution, while in the uterine sarcoma MES-SA/Dx5, another
doxorubicin-selected cell line, only the 1´-OH containing compounds showed relevant
selectivity. Since overexpression of ABCB1 did not sensitize cells, we conclude that the MDR-
selective cytotoxicity of protoflavones is connected to other changes accompanying acquired
drug resistance.
Experimental Section
Structure elucidation was carried out by means of nuclear magnetic resonance (NMR)
spectroscopy and mass spectroscopy (MS). NMR spectra were obtained on a Varian Gemini-
11
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10.1002/cmdc.201700225
ChemMedChem
2000 200 MHz or Bruker Avance DRX-500 NMR spectrometer in chloroform-d1, methanol-
d4, acetone-d6, or dimethyl sulfoxide-d6. Mass spectra were taken on an API 2000 triple-
quadrupole (Ab Sciex, USA) or LCMS-IT-TOF (Shimadzu, Japan) with an ESI interface.
Compounds were purified by rotation planar chromatography on a Chromatothron equipment
(Harrison Research, USA) with adequately chosen eluents of n-hexane-ethyl acetate on silica
GF 254 (Merc, Germany) or with Flash Chromatograpy on a Combiflash Rf+ equipment
(TELEDYNE Isco, USA) with eluents of n-hexane – ethyl acetate or methanol –
dichloromethane on RediSep normal-phase silica flash columns (TELEDYNE Isco, USA). All
compounds possessed a purity of ≥95.0% by means of HPLC-DAD, except for compounds 51
and 55 (92.20% and 93.41%, respectively), which served as intermediates for further synthesis
and whose bioactivity was not tested. All chemicals were obtained from Aldrich, Inc. (USA).
Synthesis of 5´-ethyl- and 5´-pentyl-2´-hydroxyacetophenone (41 and 42). In the first step,
4-ethyl- and 4-pentylphenol acetate (39 and 40, respectively) were synthesized by adding 0.1
mol (10.2 g) of acetic anhydride and one drop of ccH2SO4 to 0.1 mol of 4-ethyl or 4-
pentylphenol and stirring at room temperature for 20 min. The mixture was then poured into
water and extracted with 3 x 50 ml EtOAc. The organic layer was evaporated under reduced
pressure, re-dissolved in CH2Cl2 and crystallized anhydrous AlCl3 was added little by little
under ice bed cooling. The mixture was refluxed for 10 h, then the reaction was stopped by
adding crushed ice. After filtration, the precipitate was purified on silica to obtain 41 or 42.
General Procedure for chalchone synthesis. The 5´-substituted 2´-hydroxyacetophenone (41-
44) and 4-benzyloxybenzaldehyde (3.0 g, 14.3 mmol) were dissolved in 50% EtOH, KOH/H2O
solution (20 mL). The reaction was stirred at r.t. for 30 h, and then the solvent was evaporated
under reduced pressure. The mixture was purified on silica gel (isocratic elution, n-
hexane/EtOAc, 6:1) to afford 45-48, respectively.
(E)-3-(4-benzyloxyphenyl)-1-(5-ethoxy-2-hydroxyphenyl)prop-2-en-1-one (47). Orange solid;
yield: 82.2%; NP-HPLC purity: 99.15%; 1H NMR (500 MHz, chloroform-d1) δ 12.47 (1H, s,
OH), 7.89 (1H, d, J = 15.35 Hz), 7.62 (2H, d, J = 8.60 Hz), 7.47 (1H, d, J = 15.55 Hz), 7.34-
7.45 (6H, m), 7.12 (1H, dd, J = 9.00 Hz, 2.80 Hz), 7.02 (2H, d, J = 8.55 Hz), 6.96 (1H, d, J =
9.00 Hz), 5.13 (2H, s), 4.05 (2H, q, J = 6.95 Hz), 1.44 (3H, t, J = 6.95 Hz) 7.91 (2H, d, J = 8.45
Hz), 7.86 (1H, d, J = Hz), 7.67 (1H, d, J = 2.65 Hz), 7.4-7.38 (5H, br), 7.35 (1H, d, J = 7.25
Hz), 7.19 (1H, dd, J = 2.70 Hz, 8.50 Hz), 7.11 (2H, d, J = 8.50 Hz), 6.92 (1H, d, J = 9.00 Hz),
5.19 (2H, s), 4.07 (2H, q, J = 6.85 Hz), 1.34 (3H, t, J = 6.90 Hz) ; 13C NMR (125 MHz,
chloroform-d1) 193.45, 161.32, 157.97, 151.11, 145.47, 136.46, 130.69, 128.83, 128.36,
127.72, 127.61, 124.29, 119.97, 119.31, 117.93, 115.53, 114.22, 70.29, 64,72, 15.06; ESI-MS
(m/z): 375.3 [M++H].
General Procedure for Flavone synthesis. 3 mmol of chalchone (45-48) was dissolved in
DMSO (5 mL), and 1 eq. iodine (76 mg, 0.3 mmol) was added. The solution mixture was stirred
and heated to 110 °C. After 2 h, 10% Na2S2O3 (50 mL) was added to remove iodine. The
mixture was extracted with EtOAc (3 X 50 mL) and then purified by column chromatography
on silica gel to afford compounds 49-52, respectively.
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6-Ethoxy-4´-benzyloxyflavone (51). Yield: 85.2%; NP-HPLC purity: 92.20%, pale yellow
solid; 1H NMR (500 MHz, chloroform-d1) δ 7.86 (2H, d, J = 8.80 Hz), 7.56 (1H, d, J = 2.70
Hz), 7.48-7.38 (5H, benzyl), 7.35 (1H, d, J = 7.1 Hz), 7.25 (1H, dd, J = 2.85 Hz, 8.75 Hz), 7.08
(2H, d, J = 8.70 Hz), 6.72 (1H, s), 5.14 (2H, s), 4.13 (2H, q, J = 6.95 Hz), 1.44 (3H, t, J = 6.95
13
Hz); C NMR (125 MHz, chloroform-d1) 178.44, 163.26, 161.61, 156.43, 151.06, 136.34,
131.02, 128.95, 128.87, 128.41, 128.11, 127.62, 124.68, 124.61, 124.05, 119.46, 115.48,
105.72, 70.36, 64.36, 14.86; ESI-MS (m/z): 372.9 [M++H].
Synthesis of compound 53 from 52 via Suzuki-coupling. Compound 52 (407.26 mg, 1.0
mmol), 1.2 eq. phenylboronic acid (146.3 mg, 1.2 mmol) and 2 eq. (276.42mg, 2.0 mmol)
K2CO3 were dissolved in 7 mL of water, and 25 mL of propanol and 5 mmol of
Tetrakis(triphenylphosphine)palladium(0) were subsequently added. The reaction was
performed under N2 gas at 40ºC, to afford 53 as a yellow solid.
1
6-Phenyl-4´-benzyloxyflavone (53). Yield: 30 %; yellow solid; H NMR (200 MHz,
chloroform-d1) δ 8.45 (1H, s), 7.89 (3H, d, J = 7.80 Hz), 7.67 (1H, d, J = 7.20 Hz), 7.50-7.30
13
(11H, m), 7.25 (1H, s,), 7.10 (2H, d, J = 8.40 Hz), 6.77 (1H, s), 5.15 (2H, s); C NMR (50
MHz, chloroform-d1) 177.35, 162.34, 160.55, 154.56, 138.32, 137.20, 135.16, 131.35, 127.91,
127.68, 127.35, 127.21, 127.00, 126.74, 126.41, 126.12, 125.66, 123.19, 122.48, 117.39,
114.33, 113.90, 105.18, 97.23, 69.17.
General Procedure for Benzyl Group Removal. A mixture of 0.5 mmol of the 4´-
benzyloxyflavone (49-53), dry Pd/C (10%, 106 mg), and 20 mL of EtOAc was stirred at 25 °C
under an atmosphere of hydrogen for 10 h. The mixture was filtered, washed with EtOAc,
concentrated under vacuum and purified by column chromatography on silica gel to afford
compounds 54-58, respectively.
6-Pentyl-4´-hydroxyflavone (55). Yield: 85.4%; NP-HPLC purity: 93.41%, pale yellow solid;
1H NMR (500 MHz, DMSO-d6) δ 10.31 (1H, brs, OH), 7.95 (2H, d, J = 8.65 Hz), 7.81 (1H, s),
7.66 (1H, d, J = 8.45 Hz), 7.64 (1H, dd, J = 8.85 Hz, 1.85 Hz), 6.90 (2H, d, J = 9.75 Hz), 6.94
(2H, d, J = 8.60 Hz), 6.84 (1H, s), 2.70 (2H, t, J = 7.45 Hz), 1.61 (2H, quin, J = 7.00 Hz), 1.38-
1.21 (4H, m), 0.85 (2H, t, J = 6.85 Hz); 13C NMR (125 MHz, DMSO-d6) 176.97, 162.97,
160.96, 154.05, 139.67, 134.43, 128.34, 123.43, 123.07, 121.7, 118.26, 115.97, 104.72, 34.42,
30.79, 30.55, 21.96, 13.94; ESI-MS (m/z): 309.5 [M++H].
General procedure for protoflavone synthesis from 4´-hydroxyflavones. Apigenin,
genkwanin, 4´-hydroxy-6-methylflavone, 4´-hydroxy-6-methoxyflavone, 4´-hydroxy-β-
naphthoflavone (Indofine, Hillsborough, NJ, USA), or the synthesized 4´-hydroxyflavone (54-
58) was dissolved at 1 mg/mL concentration in a 9:1 v/v ratio mixture of acetonitrile and water
or the alcohol to be coupled at position C-1´. Two equivalents of
[bis(trifluoroacetoxy)iodo]benzene were added to the mixture. After stirring at 80 °C for 1 hour,
the mixture was cooled down, evaporated under reduced pressure and purified by flash
chromatography to obtain compounds 2-9,[17] 10, 11-24,[15] 25-30, 31-38,[17] or 59-73,
respectively. Compounds 10, 25-30 and 59-73 are reported here as new protoflavones.
Protoapigenone 1´-O-benzyl ether (10). Light brown solid; yield: 39,6%; RP-HPLC purity:
99.21%, 1H NMR (500 MHz, DMSO-d6) δ 12.47 (1H, s, OH), 7.36-7.43 (4H, m), 7.32 (1H, t,
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J = 6.90 Hz), 7.18 (2H, d, J = 9.95 Hz), 6.57 (2H, d, J = 9.95 Hz), 6.50 (1H, s), 6.18 (2H, d, J
13
= 9.15 Hz), 4.57 (2H, s); C NMR (125 MHz, DMSO-d6) 184.29, 181.32, 165.44, 164.37,
161.41, 157.41, 145.78, 137.53, 132.22, 128.35, 127.84, 127.65, 107.37, 103.68, 99.43, 94.01,
74.22, 66.52; ESI-MS (m/z): 377.2 [M++H].
1
6-Methoxyprotoflavone (25). Pale yellow solid; yield: 32.3%; NP-HPLC purity: 98.95%, H
NMR (500 MHz, acetone-d6) δ 7.46 (1H, d, J = 2.95 Hz), 7.44 (1H, d, J = 9.35 Hz), 7.33 (1H,
dd, J = 9.07 Hz, 2.90 Hz), 7.05 (2H, d, J = 10.00 Hz), 6.66 (1H, s), 6.33 (2H, d, J = 10.00 Hz),
6.11 (1H, s), 3.90 (3H, s); 13C NMR (125 MHz, chloroform-d1) 185.54, 179.13, 166.64, 157.4,
151.23, 147.00, 129.71, 124.57, 119.68, 107.97, 104.84, 69.85, 56.09; ESI-MS (m/z): 285.4
[M++H].
6-Methoxyprotoflavone 1´-O-methyl ether (26). Yellow solid; yield: 41.3%; NP-HPLC purity:
98.93%, 1H NMR (500 MHz, acetone-d6) δ 7.45 (1H, d, J = 3.20 Hz), 7.43 (1H, d, J = 8.7 Hz),
7.33 (1H, dd, J = 9.15 Hz, 3.00 Hz), 7.01 (2H, d, J = 10.05 Hz), 6.58 (1H, s), 6.54 (2H, d, J =
10.15 Hz), 3.90 (3H, s), 3.44 (3H, s); 13C NMR (125 MHz, DMSO-d6) 184.27, 176.53, 163.86,
156.74, 150.26, 145.91, 132.42, 123.96, 123.53, 119.89, 108, 104.77, 74.3, 55.74, 52.34; ESI-
MS (m/z): 299.1 [M++H].
6-Methoxyprotoflavone 1´-O-ethyl ether (27). Yellow solid; yield: 39.3%; NP-HPLC purity:
1
98.39%, H NMR (500 MHz, acetone-d6) δ 7.45 (1H, d, J = 2.90 Hz), 7.43 (1H, d, J = 8.85
Hz), 7.33 (1H, dd, J = 10.00, 2.80 Hz), 7.03 (2H, d, J = 10.00 Hz), 6.63 (1H, s), 6.51 (2H, d, J
13
= 9.95 Hz), 3.90 (3H, s), 3.64 (2H, q, J = 6.80 Hz) 1.28 (1H, t, J = 6.90 Hz); C NMR (125
MHz, DMSO-d6) 184.34, 176.55, 164.04, 156.73, 150.24, 146.39, 131.91, 123.96, 123.5,
119.88, 107.96, 104.78, 74.01, 60.3, 55.73, 15.56; ESI-MS (m/z): 313.0 [M++H].
6-Methoxyprotoflavone 1´-O-butyl ether (28). Yield: 46.3%; NP-HPLC purity: 98.67%, light
brown solid; 1H NMR (500 MHz, acetone-d6) δ 7.45 (1H, d, J = 2.90 Hz), 7.43 (1H, d, J = 8.80
Hz), 7.32 (1H, dd, J = 9.15 Hz, 1.80 Hz), 7.02 (2H, d, J = 10.25 Hz), 6.63 (1H, s), 6.51 (2H, d,
J = 9.85 Hz), 3.90 (3H, s), 3.59 (2H, t, J = 5.35 Hz) 1.65 (2H, q, J = 6.20 Hz), 1.51 – 1.35 (2H,
13
m), 0.94 (3H, t, J = 7.20 Hz); C NMR (125 MHz, chloroform-d1) 184.93, 178.42, 164.14,
153.28, 149.20, 146.45, 132.77, 124.66, 124.25, 119.64, 108.89, 104.99, 74.65, 65.09, 56.11,
32.23, 19.43, 13.99; ESI-MS (m/z): 341.1 [M++H].
6-Methoxyprotoflavone 1´-O-allyl ether (29). Light brown solid; yield: 37.2 %; NP-HPLC
purity: 95.17%, 1H NMR (500 MHz, acetone-d6) δ 7.44 (1H, d, J = 2.85 Hz), 7.43 (1H, d, J =
8.85 Hz), 7.30 (1H, dd, J = 9.15 Hz, 2.95 Hz), 7.02 (2H, d, J = 10.10 Hz), 6.61 (1H, s), 6.49
(2H, d, J = 9.85 Hz), 5.24 (1H, octet, J = 6.55 Hz), 4.61 (1H, d, J = 17.2 Hz), 4.43 (1H, d, J =
10.57 Hz), 3.37 (2H, d, J = 5.05 Hz), 3.86 (3H, s); 13C NMR (125 MHz, chloroform-d1) 184.69,
178.25, 163.8, 157.37, 151.09, 145.81, 145.78, 133.75, 132.87, 124.24, 123.68, 119.6, 117.69,
108.98, 105.01, 74.83, 66.23, 56.09; ESI-MS (m/z): 325.2 [M++H].
6-Methoxyprotoflavone 1´-O-proargyl ether (30). Light brown solid; yield: 37.7 %; NP-HPLC
purity: 95.32%, 1H NMR (500 MHz, acetone-d6) δ 7.46 (1H, d, J = 2.90 Hz), 7.44 (1H, d, J =
8.85 Hz), 7.33 (1H, dd, J = 9.07 Hz, 2.80 Hz), 7.10 (2H, d, J = 9.90 Hz), 6.61 (1H, s), 6.50 (2H,
13
d, J = 9.90 Hz), 4.44 (2H, d, J = 1.95 Hz), 3.90 (3H, s), 3.10 (1H, t, J = 2.20 Hz); C NMR
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ChemMedChem
(125 MHz, DMSO-d6) 184.17, 176.5, 163.24, 156.76, 150.27, 144.90, 132.26, 123.95, 123.57,
119.92, 108.17, 104.76, 80.17, 78.41, 74.40, 55.74, 53.27; ESI-MS (m/z): 322.9 [M++H].
1
6-Ethoxyprotoflavone (59). Pale yellow solid; yield: 35.3%; NP-HPLC purity: 99.14%, H
NMR (500 MHz, methanol-d4) δ 7.51 (1H, d, J = 2.90 Hz), 7.43 (1H, d, J = 9.20 Hz), 7.36 (1H,
dd, J = 9.25 Hz, 3.00 Hz), 7,00 (2H, d, J = 9.95 Hz), 6.79 (1H, s), 6.39 (2H, d, J = 10.10 Hz),
13
4.14 (2H, q, J = 6.95 Hz), 1.44 (3H, t, J = 7.00 Hz); C NMR (125 MHz, chloroform-d1)
185.37, 178.99, 166.3, 156.73, 151.08, 146.77, 129.75, 124.79, 124.23, 119.58, 107.97, 105.45,
69.78, 64.40, 14.72; ESI-MS (m/z): 299.3 [M++H].
6-Ethoxyprotoflavone 1´-O-methyl ether (60). Yellow solid; yield: 42.0%; NP-HPLC purity:
99.21%, 1H NMR (500 MHz, chloroform-d1) δ 7.48 (1H, d, J = 2.90 Hz), 7.26 (1H, d, J = 9.15
Hz), 7.20 (1H, dd, J = 9.15 Hz, 2.90 Hz), 6.79 (2H, d, J = 10.00 Hz), 6.71 (1H, s), 6.54 (2H, d,
J = 9.95 Hz), 4.09 (2H, q, J = 7.00 Hz), 3.37 (3H, s), 1.41 (3H, t, J = 7.15 Hz); 13C NMR (125
MHz, chloroform-d1) 184.64, 178.16, 163.66, 156.66, 150.94, 145.68, 133.18, 124.65, 124.47,
119.51, 108.83, 105.65, 75.01, 64.37, 52.88, 14.76; ESI-MS (m/z, %): 313.5 [M++H].
6-Ethoxyprotoflavone 1´-O-ethyl ether (61). Yellow solid; yield: 41.2%; NP-HPLC purity:
98.12%, 1H NMR (500 MHz, chloroform-d1) δ 7.49 (1H, d, J = 2.85 Hz), 7.26 (1H, d, J = 9.25
Hz), 7.20 (1H, dd, J = 9.20 Hz, 2.90 Hz), 6.80 (2H, d, J = 10.00 Hz), 6.78 (1H, s, H-3), 6.50
(2H, d, J = 10.00 Hz), 4.09 (2H, q, J = 7.00 Hz), 3.57 (2H, q, J = 6.85 Hz), 1.41 (3H, t, J = 6.85
13
Hz), 1.27 (3H, t, J = 6.95 Hz); C NMR (125 MHz, chloroform-d1) 184.83, 178.54, 164.14,
156.71, 151.02, 146.25, 132.68, 124.64, 124.53, 119.55, 108.77, 105.59, 74.71, 64.39, 61.03,
15.78, 14.75; ESI-MS (m/z, %): 327.6 [M++H].
6-Ethoxyprotoflavone 1´-O-propyl ether (62). Yellow solid; yield: 45.8%; NP-HPLC purity:
99.20%, 1H NMR (500 MHz, chloroform-d1) δ 7.49 (1H, d, J = 2.85 Hz), 7.25 (1H, d, J = 9.25
Hz), 7.20 (1H, dd, J = 9.20 Hz, 2.90 Hz), 6.79 (2H, d, J = 9.80 Hz), 6.78 (1H, s, H-3), 6.51 (2H,
d, J = 10.10 Hz), 4.09 (2H, q, J = 7.00 Hz), 3.46 (2H, t, J = 6.45 Hz), 1.65 (2H, sex, J = 7.15
Hz), 1.41 (3H, t, J = 6.90 Hz), 0.96 (3H, t, J = 7.40 Hz); 13C NMR (125 MHz, chloroform-d1)
184.85, 178.31, 163.97, 156.65, 150.95, 146.37, 132.68, 124.65, 124.45, 119.51, 108.84,
105.63, 74.59, 66.92, 64.37, 23.47, 14.77, 10.7; ESI-MS (m/z, %): 341.5 [M++H].
6-Ethoxyprotoflavone 1´-O-isopropyl ether (63). Yellow solid; yield: 43.1%; NP-HPLC purity:
98.20%, 1H NMR (500 MHz, chloroform-d1) δ 7.48 (1H, d, J = 2.75 Hz), 7.25 (1H, d, J = 9.00
Hz), 7.20 (1H, dd, J = 9.15 Hz, 2.85 Hz), 6.81 (2H, d, J = 10.00 Hz), 6.80 (1H, s), 6.48 (2H, d,
J = 9.95 Hz), 4.09 (2H, q, J = 7.00 Hz), 3.84 (2H, quin, J = 6.15 Hz), 1.42 (3H, t, J = 7.30 Hz),
1.21 (6H, d, J = 6.35 Hz); 13C NMR (125 MHz, chloroform-d1) 185.05, 178.35, 164.16, 156.64,
150.93, 146.72, 131.97, 124.64, 124.43, 119.51, 108.96, 105.62, 74.91, 68.93, 64.36, 24.82,
14.77; ESI-MS (m/z): 341.5 [M++H].
6-Ethoxyprotoflavone 1´-O-buthyl ether (64). Light brown solid; yield: 49.9%; NP-HPLC
1
purity: 98.30%, H NMR (500 MHz, chloroform-d1) δ 7.49 (1H, brs), 7.26 (1H, d, J = 9.10
Hz), 7.22 (1H, dd, J = 8.55 Hz, 2.85 Hz), 6.83 (1H, s), 6.79 (2H, d, J = 9.55 Hz), 6.51 (2H, d,
J = 9.60 Hz), 4.09 (2H, q, J = 6.85 Hz), 3.84 (2H, t, J = 6.05 Hz), 1.61 (2H, q, J = 6.65 Hz),
1.45-1.32 (2H, m), 1.41 (3H, t, J = 6.40 Hz), 0.92 (3H, t, J = 7.25 Hz); 13C NMR (125 MHz,
15
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methanol-d4) 186.46, 166.74, 158.31, 152.45, 148.05, 133.64, 125.79, 125.36, 120.82, 109.11,
106.55, 75.99, 66.07, 65.45, 33.38, 20.46, 15.07, 14.32; ESI-MS (m/z): 355.2 [M++H].
6-Ethylprotoflavone (65) Light brown solid; yield: 41.0 %; RP-HPLC purity: 95.0%, 1H NMR
(500 MHz, chloroform-d1) δ 7.92 (1H, s), 7.48 (1H, d, J = 8,55 Hz), 7.27 (1H, d, J = 8,65 Hz),
6.95 (2H, d, J = 9,30 Hz), 6.86 (1H, s), 6.38 (2H, d, J = 9,30 Hz), 2.72 (2H, q, J = 7.50 Hz),
1.25 (3H, t, J = 7.00 Hz); 13C NMR (125 MHz, chloroform-d1) 185.33, 179.31, 166.56, 154.82,
146.63, 142.31, 134.77, 129.89, 123.88, 123.37, 118.11, 108.68, 69.94, 28.44, 15.58; ESI-MS
(m/z): 283.3 [M++H].
1
6-Pentylprotoflavone (66). Light brown solid; yield: 42.3%; NP-HPLC purity: 96.10%, H
NMR (500 MHz, chloroform-d1) δ 7.92 (1H, s), 7.46 (1H, dd, J = 8.45 Hz, 1.25 Hz), 7.28 (1H,
d, J = 8.95 Hz), 6.90 (2H, d, J = 9.75 Hz), 6.79 (1H, s), 6.38 (2H, d, J = 9.80 Hz), 2,66 (2H, t,
13
J = 7.60 Hz), 1.61 (2H, quin, J = 6.90 Hz), 1.38-1.21 (4H, m), 0.85 (3H, t, J = 7.05 Hz); C
NMR (125.7 MHz, chloroform-d1) 176.91, 162.93, 160.90, 154.00, 139.61, 134.37, 123.38,
118.20, 104.68, 34.37, 30.48, 20.73, 21.90, 13.88; ESI-MS (m/z): 325.4 [M++H].
1
6-Phenylprotoflavone (68). Yellow solid; yield: 38.1%; NP-HPLC purity: 95.19%, H NMR
(500 MHz, chloroform-d1) δ 8.37 (1H, d, J = 1.40 Hz), 7.88 (1H, dd, J = 9.00 Hz, 1.85 Hz),
7.62 (2H, d, J = 7.55 Hz), 7.45 (2H, t, J = 6.75 Hz), 7.41 (1H, d, J = 8.95 Hz), 7.37 (1H, t, J =
13
7.65 Hz), 6.91 (2H, d, J = 10.10 Hz), 6.82 (1H, s), 6.57 (2H, d, J = 9.90 Hz); C NMR (50
MHz, chloroform-d1) δ 145.07, 131.64, 128.27, 127.94, 127.42, 126.95, 126.10, 122.51,
122.51, 117.47, 108.50, 65.77; HRESIMS C21H15O4, calcd. 331.0970, found: 331.0973.
6-Phenylprotoflavone 1´-O-methyl ether (69). Yellow solid; yield: 50.2%; NP-HPLC purity:
1
97.74%, H NMR (500 MHz, chloroform-d1) δ 8.37 (1H, d, J = 1.50 Hz), 7.88 (1H, dd, J =
8.35 Hz, 1.50 Hz), 7.66 (2H, d, J = 7.85 Hz), 7.45 (2H, t, J = 7,10 Hz), 7.41 (1H, d, J = 8.85
Hz), 7.37 (1H, t, J = 7.00 Hz), 6.82 (2H, d, J = 9.95 Hz), 6.77 (1H, s), 6.57 (2H, d, J = 9.90
Hz), 3.41 (3H, s); 13C NMR (50 MHz, chloroform-d1) δ 144.39, 132.14, 131.81, 128.26,
127.94, 127.42, 127.26, 126.92, 126.10, 122.51, 121.72, 117.47, 108.49, 108.11, 51.71, 28.63;
HRESIMS C22H17O4, calcd. 345.1127, found: 345.1125.
6-Phenylprotoflavone 1´-O-ethyl ether (70). Yellow solid; yield: 48.2%; NP-HPLC purity:
98.10%, 1H NMR (500 MHz, chloroform-d1) δ 8.37 (1H, d, J = 1.50 Hz), 7.87 (1H, d, J = 8.60
Hz), 7.62 (2H, d, J = 7.50 Hz), 7.44 (2H, t, J = 7.20 Hz), 7.40 (1H, d, J = 8.85 Hz), 7.36 (1H,
brt, J = 6.75 Hz), 6.84 (1H, s), 6.83 (2H, d, J = 10.10 Hz), 6.53 (2H, d, J = 9.80 Hz), 3.58 (2H,
q, J = 7.00 Hz), 1.28 (3H, t, J = 6.80 Hz); 13C NMR (50 MHz, chloroform-d1) δ 144.84, 132.63,
131.78, 128.29, 127.96, 127.42, 127.24, 126.91, 126.10, 122.51, 121.70, 117.49, 108.52,
108.15, 59.85, 28.63, 14.60; HRESIMS C23H19O4, calcd. 359.1283, found: 359.1280.
6-Phenylprotoflavone 1´-O-propyl ether (71). Light brown solid; yield: 46.4%; NP-HPLC
purity: 99.39%, 1H NMR (500 MHz, chloroform-d1) δ 8.37 (1H, d, J = 1.40 Hz), 7.88 (1H, dd,
J = 8.85 Hz, 1.60 Hz), 7.62 (2H, d, J = 7.50 Hz), 7.44 (2H, brt, J = 6.45 Hz), 7.40 (1H, d, J =
8.70 Hz), 7.36 (1H, t, J = 7.20 Hz), 6.84 (1H, s), 6.83 (2H, d, J = 9.95 Hz), 6.54 (2H, d, J =
9.90 Hz), 3.48 (2H, t, J = 6.25 Hz), 1.67 (2H, sex, J = 6.95 Hz), 0.98 (3H, t, J = 7.35 Hz); 13C
NMR (50 MHz, chloroform-d1) δ 145.07, 131.64, 128.27, 127.94, 127.42, 126.95, 126.10,
16
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ChemMedChem
122.51, 117.47, 108.50, 65.77, 30.89, 28.63, 22.27, 9.51; HRESIMS C24H21O4, calcd.
373.1440, found: 373.1438.
6-Phenylprotoflavone 1´-O-isopropyl ether (72). Light brown solid; yield: 42.2%; NP-HPLC
1
purity: 99.30%, H NMR (500 MHz, chloroform-d1) δ 8.37 (1H, brs), 7.88 (1H, dd, J = 8.10
Hz, 1.25 Hz), 7.62 (2H, d, J = 7.85 Hz), 7.45 (2H, brt, J = 6.65 Hz), 7.41 (1H, d, J = 8.85 Hz),
7.37 (1H, t, J = 7.00 Hz), 6.82 (2H, d, J = 9.95 Hz), 6.77 (1H, s), 6.57 (2H, d, J = 9.90 Hz),3,89
(1H, sext, J = 6.25 Hz); 13C NMR (50 MHz, chloroform-d1) δ 145.44, 138.05, 132.61, 128.30,
127.96, 127.44, 127.24, 126.91, 126.10, 122.51, 121.70, 117.47, 108.62, 108.26, 97.24, 67.81,
28.63, 23.62; HRESIMS C24H21O4, calcd. 373.1440, found: 373.1439.
6-Phenylprotoflavone 1´-O-butyl ether (73). Light brown solid; yield: 50.1%; NP-HPLC purity:
95.4%; 1H NMR (200 MHz, chloroform-d1) δ 8.40 (1H, d, J = 1.50 Hz), 7.89 (1H, dd, J = 9.02
Hz, 2.00 Hz), 7.64 (2H, d, J = 8.00 Hz), 7.47 (2H, brt, J = 6.60 Hz), 7.41 (1H, d, J = 8.20 Hz),
7.39 (1H, t, J = 7.00 Hz), 6.81 (1H, s), 6.56 (2H, d, J = 10.00 Hz), 6.39 (2H, d, J = 9.80 Hz),
13
3.54 (2H, t, J = 5.80 Hz), 1.68-1.36 (4H, m), 0.95 (3H, t, J = 7.20 Hz); C NMR (50 MHz,
chloroform-d1) δ 145.01, 138.03, 132.63, 128.27, 127.96, 127.42, 127.24, 126.91, 126.10,
122.52, 121.72, 117.47, 108.52, 108.15, 63.89, 31.01, 28.63, 18.20; HRESIMS C25H23O4,
calcd. 387.1596, found: 387.1593.
Cell lines. L5178 mouse T-cell lymphoma cell line (ECACC catalog no. 87111908, U.S. FDA,
Silver Spring, MD, U.S.), and its sub-cell line L5178B1, derived from L5178 by transfection
with pHa MDR1/A retrovirus,[26] were cultured in McCoy’s 5A media supplemented
inactivated horse serum. L5178B1 cell line was selected by culturing the infected cells with 60
μg/L colchicine (Sigma). Breast cancer cell lines MCF7 and its sub-cell line obtained by
adaptation to doxorubicin, MCF7Dox[27] were cultured in EMEM media supplemented with non-
essential amino acids, 1mM Na-pyruvate and 10% inactivated fetal bovine serum (MCF7Dox
was cultured in presence of 1 μM of doxorubicin each third passage). All above cell lines were
cultured at 37°C and 5% CO2; all media contained Nystatin, 2 mM of L-glutamine, 100U of
penicillin and 0.1mg of streptomycin, purchased from Sigma.
MES-SA human uterine sarcoma cell line and the doxorubicin selected MES-SA/Dx5 were
obtained from ATCC. The human cervix carcinoma cell line KB-3-1 and its vinblastine selected
derivative KB-V1 were a kind gift from Dr. Michael M. Gottesman (National Institutes of
Health). A431 and the retrovirally transduced A431B1 and A431G2 are human skin-derived,
epidermoid carcinoma cells were kind gifts from Dr. K. Német. MES-SA, KB-3-1, A431 and
their derivative cell lines were maintained in DMEM completed with 10% FBS, 5 mM
glutamine and 50 units/mL penicillin and streptomycin (Life Technologies).
Cytotoxicity assay. In case of mouse lymphoma cell lines L5178 and L5178B1, 2x104 cells per
well were cultured in 96-wells microplates with different concentrations of the tested
compound, in McCoy’s 5A media, at 37ºC and 5% CO2, for 24h.
With respect to MCF7 and MCF7Dox, 1x104 cells per well were seeded overnight and serial
dilutions of the compounds were added the following day and incubated for 48h. In all cases,
after the incubation time, 10% MTT was added to each well and incubated for 4h, when 100 μM
of 10% sodium dodecyl-sulfate (SDS) dissolved in 0.01M HCl was added to each well. Results
were read after o/n incubation. Fifty per cent inhibitory concentrations (IC50) were calculated
17
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10.1002/cmdc.201700225
ChemMedChem
using nonlinear regression curve fitting of log (inhibitor) versus normalized response with a
variable slope and least squares (ordinary) fit of GraphPad Prism 5 software, for three
independent samples.
In the case of A431 cell lines, MES-SA cell lines, KB-3-1 and KB-V1 cell lines, 5x103 cells
per well were cultured and incubated overnight in 96-well microplates. Serially diluted drugs
were then added, and plates were incubated for additional 72 hours. Cytotoxicity was measured
by Presto Blue cell viability reagent (Invitrogen) in a final dilution of 5%.
FACS measurements. Calcein accumulation assay was performed as described earlier[21]
.
Briefly, 250 000 cells per tube were pre-incubated for 5 minutes in the presence of 20 µM
verapamil or the test protoflavone compounds. Calcein AM was added at a final concentration
of 250 nM and incubated for an additional 10 minutes. Samples were then washed with ice-
cold PBS and were kept on ice until measured by an Attune® Acoustic Focusing Cytometer.
Supporting information available: Cytotoxicity data on the studied cancer cell line pairs and
corresponding selectivity ratios, as well as data of the calcein accumulation assay are presented
as supplementary Tables S1-S4, and correlation of IC50 values on the L5178B1, MCF-7 and
MCF-7Dox cell lines with those on the L5178 cells as Figure S1.
Acknowledgements
This work was supported by the National Research, Development and Innovation Office,
Hungary (NKFIH; K119770). A.H. acknowledges the János Bolyai fellowship of the Hungarian
Academy of Sciences, the Kálmán Szász Prize and Networking contribution from COST
Actions CM1106 (Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells)
and CM1407 (Challenging organic syntheses inspired by nature—from natural products
chemistry to drug discovery). G.S. was supported by ERC (StG-260572), TÉT_13_DST-1-
2013-0012 and a Momentum Grant of the Hungarian Academy of Sciences. Support from the
National Science Council (NSC), Taiwan and the Szeged Foundation for Cancer Research, as
well as a bilateral mobility grant from the Hungarian Academy of Sciences and the NSC Taiwan
is acknowledged. B.D. acknowledges Zoltán Ujlaki for his efforts in synthesizing compounds
59-64 and Dr. Da-Wei Chuang (Graduate Institute of Natural Products, KMU, Kaohsiung,
Taiwan) for scientific discussion.
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Table of Contents Graphic
ABCB1 or ABCG2 TRANSFECTED
MDR
cancer cell
Parental
cancer cell
Evasion of
resistance
Kills both with similar efficacy
ADAPTED to chemotherapeutics
R: Me, Et,
Pr, i-Pr,
Bu, Allyl,
Propargyl,
Bn
MDR
Selective
cytotoxicity
Parental
R1: Me, Et, Pent,
OMe, OEt, Ph
cancer cell
cancer cell
Can preferably kill resistant cell
20
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