Triazole Bridged Flavonoid Dimers as Potent, Nontoxic, and Highly Selective Breast Cancer Resistance Protein (BCRP/ABCG2) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b00963

The present work describes the syntheses of diverse triazole bridged flavonoid dimers and identifies potent, nontoxic, and highly selective BCRP inhibitors. A homodimer, Ac22(Az8)₂, with m-methoxycarbonylbenzyloxy substitution at C-3 of the flavone moieties and a bis-triazole-containing linker (21 atoms between the two flavones) showed low toxicity (IC₅₀ toward L929, 3T3, and HFF-1 > 100 μM), potent BCRP-inhibitory activity (EC₅₀ = 1-2 nM), and high BCRP selectivity (BCRP selectivity over MRP1 and P-gp > 455-909). Ac22(Az8)₂ inhibits BCRP-ATPase activity, blocks the drug efflux activity of BCRP, elevates the intracellular drug accumulation, and finally restores the drug sensitivity of BCRP-overexpressing cells. It does not down-regulate the surface BCRP protein expression to enhance the drug retention. Therefore, Ac22(Az8)₂ and similar flavonoid dimers appear to be promising candidates for further development into combination therapy to overcome MDR cancers with BCRP overexpression.

Full text of DOI:10.1021/acs.jmedchem.9b00963

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Article
Triazole Bridged Flavonoid Dimers as Potent, Nontoxic and Highly
Selective Breast Cancer Resistance Protein (BCRP/ABCG2) Inhibitors
Xuezhen Zhu, Iris L. K. Wong, Kin Fai Chan, Jiahua Cui, Man Chun Law,
Tsz Cheung Chong, Xuesen Hu, Larry M C CHOW, and Tak-Hang Chan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00963 • Publication Date (Web): 29 Aug 2019
Downloaded from pubs.acs.org on August 30, 2019
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Triazole Bridged Flavonoid Dimers as Potent, Nontoxic and Highly Selective Breast Cancer
Resistance Protein (BCRP/ABCG2) Inhibitors
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Xuezhen Zhu , Iris L.K. Wong , Kin-Fai Chan , Jiahua Cui Man Chun Law , Tsz Cheung
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Chong , Xuesen Hu , Larry M.C. Chow* and Tak Hang Chan*
1Department of Applied Biology and Chemical Technology and State Key Laboratory of
Chemical Biology and Drug Discovery, Hong Kong Polytechnic University, Hong Kong SAR,
China
2School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai
200240, China
3Department of Chemistry, McGill University, Montreal, Quebec, H3A 2K6 Canada
#These authors contributed equally to this work.
*Co-corresponding authors: Tak Hang Chan and Larry M. C. Chow
For L. M. C. C., Phone: (852)-34008662: Fax: (852)-23649932; E-mail:
larry.chow@polyu.edu.hk
For T. H. C., Phone: (852)-34008670; Fax: (852)-23649932; E-mail: tak-hang.chan@polyu.edu.hk
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ABSTRACT
The present work describes the syntheses of diverse triazole bridged flavonoid dimers and
identifies potent, nontoxic and highly selective BCRP inhibitors. A homodimer, Ac22(Az8) , with
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m-methoxycarbonylbenzyloxy substitution at C-3 of the flavone moieties and a bis-triazole-
containing linker (21 atoms between the two flavones) showed low toxicity (IC towards L929,
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T3 and HFF-1 > 100 µM), potent BCRP-inhibitory activity (EC = 1-2 nM) and high BCRP
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selectivity (BCRP selectivity over MRP1 and P-gp > 455-909). Ac22(Az8) inhibits BCRP-
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ATPase activity, blocks the drug efflux activity of BCRP, elevates the intracellular drug
accumulation, and finally restores the drug sensitivity of BCRP-overexpressing cells. It does not
down-regulate the surface BCRP protein expression to enhance the drug retention. Therefore,
Ac22(Az8) and similar flavonoid dimers appear to be promising candidates for further
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development into combination therapy to overcome MDR cancers with BCRP overexpression.
Keywords: CuAAC reaction, multidrug resistance, breast cancer resistance transporter, BCRP
modulators, flavonoids
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1
. INTRODUCTION
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Cancer causes significant number of human deaths in the world. For cancer patients,
chemotherapy remains one of the critical treatments. However, multidrug resistance (MDR) often
emerges to become a major impediment to successful chemotherapy. Overexpression of an active
ATP-binding cassette (ABC) transporter protein on the plasma membrane of cancer cells is a
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common mechanism for causing drug resistance. Three major ABC transporter proteins including
P-glycoprotein (P-gp; ABCB1), multidrug resistance-associated protein 1 (MRP1; ABCC1), and
breast cancer resistance protein (BCRP; ABCG2) have been associated with MDR in cancer.3-6
They can pump many structurally and mechanistically diverse anticancer drugs out of the cancer
cells and render these drugs ineffective at a therapeutic dosage. Using the mutliplatform approach
analysis
(https://www.carislifesciences.com/wp-content/uploads/2015/05/Drug-Efflux-Pump-
Expression-in-50000-Molecularly-Profiled-Cancer-Patients_ASCO-2015.pdf), across all tumors
profiled (n=51,939), MRP1 positivity was highest at 81%, BCRP at 66% and P-gp the lowest at
23%. About 29% of the patients tested exhibits co-expression of all three drug transporters. The
ABC transporter proteins are also expressed in many normal tissues and are believed to perform a
protective role. For ABC transporters expressed in the brain, testis and placenta, they protect these
‘sanctuaries’ from cytotoxins; and for those expressed in the liver, gastrointestinal tract and kidney
they are used to excrete toxins, protecting the entire organism.7
BCRP, unlike P-gp and MRP1, is a 72kDa half transporter and has a distinct structure which
consists of only one cytosolic nucleotide-binding domain (NBD) and one transmembrane domain
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(TMD). Several investigations have indicated that it would form a dimeric or tetrameric structure
when it functions as a transporter.9-11,12, 13 BCRP was found to be overexpressed in a number of
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human cancers and associated with clinical drug resistance and lower survival rate.14-21 Moreover,
it is highly overexpressed in normal and putative cancer stem cells.22-24 Since its identification in
1998,25-27 numerous anticancer drugs including methotrexate,28 mitoxantrone,29 topotecan,30
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irinotecan and its active metabolite SN-38, as well as some tyrosine kinase inhibitors (TKIs),
have been identified as substrates of BCRP. Co-administration of potent inhibitor of ABC
transporter such as P-gp with an anticancer drug has been evaluated in several clinical trials to
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overcome MDR but led to disappointing outcome. One factor which may account for the lack of
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success is likely the effect of the inhibitor on the pharmacokinetics of the anticancer drug. It is
suggested that further improvement of inhibitors of ABC transporters should focus on potency and
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specificity to minimize unexpected pharmacokinetic effects. In the case of BCRP, the first
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identified inhibitor was fumitremorgin C (Figure 1) with EC round 1 - 5 µM.
It was found
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to be neurotoxic and unsuitable for clinical development. In subsequent studies, one of the
analogues of fumitremorgin C, Ko143 (Figure 1)37 was identified as a potent and selective
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inhibitor of BCRP with EC around 10 nM. It is not stable in mouse plasma, even though when
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topotecan in mice. Tariquidar (Figure 1), a third-generation P-gp inhibitor, has also been
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reported to inhibit BCRP with EC around 100 nM. Recently, Wiese and his group reported that
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,4,6-substituted quinazolines (with EC = 20 - 71 nM), 2,4-disubstituted pyridopyrimidines
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with EC = 37 nM) and 4-anilino-2-pyridylquinazolines and -pyrimidines (with EC = 21 nM),
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are highly potent and nontoxic inhibitors of BCRP. Recently, the crystal structure of human
BCRP has been reported and provides insight about the binding of BCRP with its substrates and
inhibitors.45, 46 These binding results may provide the essential structural basis for discovery of
novel BCRP modulators.
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Figure 1. Chemical structures of fumitremorgin C, Ko143, tariquidar and general structure I &
II.
Flavonoids are abundantly present in fruits and vegetables and commonly regarded as safe
substances for human consumption. Many natural flavonoids are found to exhibit moderate activity
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in modulating ABC transporters. Because of the pseudo-dimeric structure of ABC transporters,
we reasoned that a bivalent approach by coupling two flavonoid moieties together may provide
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potent and non-toxic inhibitors of ABC transporters. Previously, we synthesized a series of
flavonoid dimers of general structure I (Figure 1) with polyethylene glycol (PEG) linkers and
some of them showed promising P-gp- and MRP1-modulating activities with nanomolar EC50
values (70 to 170 nM).48-53 The inhibitory selectivity of I towards P-gp or MRP-1 depends critically
on the linker length, with four PEG groups in I (n=4) selective for P-gp48-51 and with five to six
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PEG groups (I, n=5-6) selective for MRP1. Recently, we have successfully employed the copper
I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction between azide and alkyne to efficiently
synthesize triazole-linked flavonoid dimers of general structure II (Figure 1) as potent MRP1
(
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inhibitors. Interestingly, out of the 300-member synthetic library, the 21 most active compounds
(EC = 53-298 nM for reversing DOX resistance in 2008/MRP1) discovered in these triazole-
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similar in linker length to I with n=5-6. In the process, we also found that some of the compounds
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showed inhibitory activities against BCRP. In this study, we further construct more triazole-
linked flavonoid dimers to discover potent and nontoxic inhibitors of BCRP, to explore the
structure activity relationship and, on that basis, discover specific inhibitor of BCRP over P-gp
and MRP1.
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. RESULTS
.1. Design and synthesis of triazole and bis-triazole bridged flavonoid dimers
The copper(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction between azides and
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alkynes (commonly known as CuAAC reaction) was used to prepare the triazole and bis-triazole
bridged flavonoid dimers. The alkynes Ac1-3, Ac5, Ac12, Ac13 and Ac16 and the azides Az1-3,
Az11-13 and Az17 were conveniently prepared according to the procedure described in the
previous study.54 To further expand structural diversity, we synthesized additional flavonoid
bearing azides Az8, Az9, Az14 and Az15. The azides Az8-9 were prepared starting from
compounds 1a-b which was followed by debenzylation and alkylation with methyl 3-
(bromomethyl)benzoate to furnish compounds 1c-d. Azides Az8-9 were realized after the
conversion of the hydroxyl group in 1c-d to azido group (Scheme 1). For Az14-15, the
hydroxylated flavone 2b-c were coupled with 2,2'-(benzylazanediyl)diethanol under Mitsunobu
condition and the hydroxyl group of the coupling product was converted to azido group to furnish
the desired azides (Scheme 2). With one flavonoid bearing an acetylene group AcN and another
flavonoid bearing an azido group AzM, a triazole bridged flavonoid dimer AcNAzM could be
easily obtained by employing the CuAAC reaction. (Scheme 3). Treatment of the diacetylenes
Ac15 with two mole equivalents of azides (Az1-3, Az8, Az9, Az11-13, Az16 and Az17) in the
presence of catalytic amount of Cu(PPh ) Br under THF refluxing temperature afforded the bis-
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triazole bridged flavonoid dimers Ac15(AzM) (Scheme 3). Their dimeric nature was confirmed
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by their high-resolution mass and NMR spectra. Altogether, a library of 74-member of pure
triazole and bis-triazole bridged homo- and hetero-flavonoid dimers was constructed and tested
with biological assays for inhibitory activity against BCRP, MRP1 and P-gp.
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After biological assays of these flavonoid dimers, Ac15(Az8) and Ac15(Az9) exhibited not
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only high BCRP-modulating activity but also good selectivity for BCRP. These promising results
encouraged us to carry out further structural optimization of these compounds with various
diacetylenes including Ac22-31. The diacetylenes Ac22-25 are commercially available. The
diacetylenes Ac26, Ac29 and Ac31 were prepared according to the previous procedure.54-56
Treatment of amine 3a-b with propargyl bromide furnished the diacetylenes Ac28 and Ac30
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Scheme 4). The desired bis-triazole bridged flavonoid dimers were furnished by treatment of
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in reasonably good yields (Scheme 5). The chemical structures of all alkynes, azides and clicked
dimers were summarized in Figures 2 and 3 and Table 1, respectively.
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Ac1 R = H, n=3
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Figure 2. Structures of alkynes.
Azide-bearing flavonoids
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Az1 R = H, n = 1
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Az3 R = 6-Me, n = 2
Az4 R = 6-F, n = 2
Az5 R = 3-OBn, n =2
Az7 R = 6-F, n = 1
Az10 R = 3-OBn, n = 1
Az11 n = 1
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Figure 3. Structures of azides.
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Scheme 1. Synthesis of azides Az8 and Az9.a
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O
(
ii)
O
(i)
O
O
OH
N3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
O
O
n
O
n
O
O
O
n
1
1
c, n=1
d, n=2
Az8, n=1
Az9, n=2
1a, n=1
1b, n=2
Yield: 1c,86%
Yield: Az8,57%
Az9,37%
1
d,62%
a
Reagents and condition: (i) (a) H , Pd/C, MeOH, rt; (b) K CO , methyl 3-(bromomethyl)benzoate,
2
2
3
o
acetone, reflux; (ii) (a) methanesulfonyl chloride, NEt , DCM, 0 C; (b) NaN , ACN.
3
3
Scheme 2. Synthesis of azides Az14 and Az15.a
O
O
O
N
O
O
N
3
OH
Az14
2
b
Yield:17%
(i)
+
O
(
ii)
N
HO
OH
O
O
2a
O
OH
N
O
N
3
2
c
Az15
Yield:22%
a
Reagents and condition: (i) PPh , DIAD, THF; (ii) (a) methanesulfonyl chloride, NEt , DCM,
3
3
o
0 C; (b) NaN , ACN.
3
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Scheme 3. Synthesis of triazole and bis-triazole bridged flavonoid dimers a
Az
(i)
Ac
+
Az
N
N
Ac
N3
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ac2Az2
Ac1-3,5,12,13,15,16
Az1-5,7-15
:
:
Ac16Az13
Yield: 24%-98%
Bn
A
z
Az
Az
(i)
+
N
N
N3
N
N
N
N
N
N
Bn
Ac15
Az1-3,5,8,9,11-13,16,17
Ac15(Az1)2
:
:
Ac15(Az17)2
Yield: 49%-99%
a
Reagents and condition: (i) cat. Cu(PPh ) Br, THF, reflux, 12hr.
3
3
Scheme 4. Synthesis of diacetylenes Ac28 and Ac30 a
NH2 Br
N
N
N
(
i)
3
3
a
Ac28
Yield: 38%
Br
NH
N
N
HN
(i)
b
Ac30
Yield: 55%
a
Reagents and condition: (i) propargyl bromide, acetone, rt.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 5. Synthesis of analogues of Ac15(Az8) and Ac15(Az9) .
2
2
Yield
N
N
N
N
Az
Az
Az
Az
N
N
Ac22(Az8) , 75%
Ac22(Az9) , 65%
2
2
Ac22
Ac23
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ac23(Az8) , 65%
Ac23(Az9) , 65%
2
N
N
N
2
N
N
N
N
N
N
N
Ac24(Az8)2, 65%
Ac25(Az8)2, 57%
Az
Az
N
N
Az
Ac24
O
N
N
N
N
N
N
Az
Ac25
O
N
Ac26(Az8) , 58%
Ac26(Az9) , 55%
2
Az
(i)
Az
Az
N
N
N
N
+
N
Az
Az
N3
2
N
N
Az8, Az9
Ac26
Ac28(Az8) , 54%
N
N
N
2
N
O
O
N
N
N
N
O
N
Az
=
O
Ac28
N
N
O
Ac29(Az8) , 52%
Ac29(Az9) , 45%
2
Az
Az
N
O
n
N
N
N
N
2
O
N
N
N
N
N
Az8 n =1
Az9 n =2
N
N
N
Ac29
Az
Ac30(Az8) , 59%
N
Az
Az
2
N
N
N
N
N
N
N
Ac30
Ac31(Az8) , 69%
2
Az
N
N
N
N
N
N
N
N
Ac31
a
Reagents and condition: (i) cat. Cu(PPh ) Br, THF, reflux, 12hr.
3
3
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2
2
.2. Biological assay results
.2.1. BCRP-modulating activity of triazole and bis-triazole bridged flavonoid dimers
For the biological assays, four different cell lines were employed in this study including: (i)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BCRP-transfected human embryonic kidney cell line HEK293/R2; (ii) mitoxantrone-selected
breast cancer cell line MCF7-MX100 in which the BCRP transporter protein was found to be
overexpressed; (iii) MRP1-transfected ovarian cancer cell line 2008/MRP1 and (iv) P-gp-
transfected human breast cancer cell line LCC6MDR (Table 1). Effective concentration (EC ), at
5
0
which the modulator can reduce the IC of an anticancer drug towards a cell line by half, was used
5
0
to differentiate the inhibitory potency of the flavonoid dimers. A lower EC value implies that the
50
flavonoid dimer displays higher potency to reverse BCRP-, MRP1- and P-gp-mediated drug
resistance. For positive controls in the biological assays, Ko143 (EC = 9 nM in HEK293/R2 and
5
0
MCF7-MX100), a BCRP-specific modulator, cyclosporine A (EC = 32 nM in LCC6MDR), a
5
0
known P-gp inhibitor, and the flavonoid dimer, FD-4e (Figure 1, EC = 111 nM in 2008/MRP1),
5
0
52
previously reported to possess promising MRP1-modulating activity were used. The triazole
bridged flavonoid dimers would be considered as potent MDR chemosensitizers if they exhibit
EC values lower than that of positive controls. In addition, the cytotoxicity (IC ) of these
50
50
compounds towards L929 cells, a normal mouse fibroblast cell line, was also determined as an
indication of their potential toxicity. The results are summarized in Table 1.
Some of these flavonoid dimers exhibited potent MDR inhibitory activity against BCRP
for the two cell lines HEK293/R2 and MCF7-MX100 in restoring topotecan cytotoxicity (Table
1
). As reported in Table 1, among the 74 compounds tested, 11 compounds, Ac2Az9, Ac3Az4,
Ac3Az9, Ac3Az11, Ac5Az8, Ac5Az9, Ac12Az8, Ac12Az9, Ac13Az9, Ac15(Az8) and
2
Ac15(Az9) , display a more potent activity than Ko143 in both cell lines with EC in the range of
2
50
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
.9-7.9 nM. Equally important is the fact that these potent compounds are essentially nontoxic
towards L929 with IC >100 µM. In contrast, Ko143 has an IC = 31 µM. These triazole bridged
5
0
50
flavonoid dimers are therefore potent and nontoxic inhibitors of BCRP.
.2.2. Structure Activity Relationship (SAR) of flavonoid dimers and BCRP-inhibitory
activity
In order to understand the SAR for inhibiting BCRP activity, we have (1) introduced various
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
substituents at C-6 or C-7 on A-ring or at C-3 on C-ring of the flavonoid moiety; (2) synthesized
different linker length between the two flavonoids; (3) located the linker at different positions of
the flavonoid; and (4) designed the linker with a N-atom bearing benzyl substituent. For easier
analysis of the results, we reformatted the EC data of Table 1 into Table 2A and 2B. Each cell
5
0
in Table 2 represents the EC of AcNAzM in reversing topotecan resistance in HEK293/R2
5
0
(Table 2A) or MCF7-MX100 (Table 2B) with different color coding for different potency. First,
the potency is attributed to the presence of the two flavonoid moieties. The compound
Ac15(Az16) contains no flavonoid and is essentially nonactive (EC >1000 nM) whereas all other
2
50
compounds in Table 2 with two flavonoid moieties are active (EC 1-543 nM). Secondly, it is
5
0
clear from Table 2 that flavonoid dimers derived from Az8 or Az9 are highly potent irrespective
of the Ac component to which they are coupled with EC ranged from 1 to 24 nM. For dimers not
5
0
containing Az8 or Az9, the most potent compound is Ac3Az4 with EC = 3.1- 4.0 nM, suggesting
5
0
that a fluorine substitution in ring A of the azide component can enhance potency. Comparing Az2
with Az12 where the difference is the location of the linker, suggesting that placing the linker at
C-7 of the A ring in the azide component may be preferred. On the other hand, replacing an oxygen
atom in the linker by a benzylamine group did not lead to lower EC as we compare Ac5Az14
5
0
with Ac5Az1 or Ac5Az15 with Ac5Az11.
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Among the different acetylene-derived dimers, Ac3AzM dimers displayed the most
promising BCRP inhibitory potency if we exclude the dominant effect of Az8 and Az9 on activity
mentioned above (Table 2A and 2B), suggesting that 7-fluoro substitution on A-ring in acetylene-
containing flavone may play an important role in enhancing the BCRP inhibitory activity. In
contrast, a 6-methyl substitution (Ac5AzM) on A-ring did not increase the potency as the EC50
ranged from 15 nM to 543 nM, excluding Ac5Az8 and Ac5Az9. Comparing Ac1Az1 with
Ac2Az1, their EC values are similar, suggesting that the location of the linker for the Ac
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
component, either at A or B ring, does not make a substantial difference on the activity.
Ac15 is different from all other AcN considered thus far. It is a diacetylene with no flavonoid
by itself. It generated flavonoid homodimers when it was coupled with 2 mole equivalents of azido
monomers, AzM, to form Ac15(AzM) . Their BCRP-modulating activity appear to be similar to
2
other flavonoid dimers AcNAzM (Table 2). However structurally, Ac15(AzM) contain two
2
triazole groups in the linker whereas the other AcNAzM dimers have only one triazole group in
the linker. The effect of such structural difference on MRP1- and P-gp-modulating activity would
be further explored.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1. BCRP-, MRP1- and P-gp-modulating activity of flavonoid dimers.
F1
F2
EC50 (nM) for reversing MDR
BCRP-mediated MRP1-mediated
topotecan resistance DOX resistance paclitaxel resistance
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
P-gp mediated
Compounds
N
N
F2
N
O
F1
O
x y
y
O
x
MCF7-MX100
2008/MRP1
LCC6MDR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
O
O
Ac1Az1*
Ac2Az1*
Ac2Az2
Ac2Az3
Ac2Az4*
Ac2Az5
3 1
60.2 ± 16.3*
12.3 ± 6.7
66.7 ± 7.6
51.7 ± 20.2
34.7 ± 18.2
38.3 ± 32.8
23.1 ± 15.2
127.5 ± 32.5*
201.0 ± 1.4
O
O
O
3 1 >100*
29.5 ± 26.5
202.7 ± 101.3
185.0 ± 7.1
97.5 ± 59.4
151.3 ± 14.2*
170.0 ± 17.3
200.0 ± 62.4
81.4 ± 8.5*
300.0 ± 113.1
384.0 ± 15.1
349.5 ± 74.2
328.0 ± 87.6
O
O
3 2
3 2
12.0 ± 6.2
O
O
>50
O
O
F
3 2 >100*
O
O
O
O
O
O
3 2 >100
3 1 >100
3 2 >100
3 1 >100*
59.7 ± 9.0
7.2 ± 3.1
78.3 ± 1.5
23.6 ± 29.2
7.0 ± 5.0
207.3 ± 63.1
212.0 ± 69.3
181.4 ± 43.8
297.8 ± 44.3*
205.0 ± 55.2
258.8 ± 77.2
616.3 ± 167.2
414.3 ± 168.8
489.7 ± 61.8
515.0 ± 106.1
O
O
O
O
Ac2Az8
Ac2Az9
O
O
O
O
3.5 ± 2.0
O
O
O
Ac2Az11*
Ac2Az12
42.0 ± 15.6
57.0 ± 8.5
22.3 ± 43.2
176.7 ± 86.2
O
O
3 2
>50
O
1
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Table 1…………….continued
F1
F2
EC50 (nM) for reversing MDR
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
BCRP-mediated
topotecan resistance
MCF7-MX100
MRP1-mediated
P-gp mediated
paclitaxel resistance
LCC6MDR
N
F2
N
Compounds
O
F1
N
O
DOX resistance
2008/MRP1
x y
y
O
x
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
O
O
Ac3Az1*
Ac3Az2*
Ac3Az3*
Ac3Az4*
Ac3Az5
3 1 >100*
3 2 >100*
3 2 >100*
3 2 >100*
3 2 >100
3 1 >100
3 2 >100
3 1 >100*
3 2 >100
7.2 ± 4.4
24.0 ± 15.6
14.0 ± 1.4
3.1 ± 2.1
17.5 ± 11.4
7.4 ± 8.0
137.0 ± 16.2*
208.0 ± 48.1*
114.5 ± 10.9*
131.7 ± 19.8*
111.0 ± 13.9
137.0 ± 7.2
155.0 ± 56.6
255.0 ± 9.9
O
O
O
19.7 ± 17.4
4.0 ± 3.2
218.5 ± 99.7
161.0 ± 50.9
344.4 ± 91.8
480.7 ± 183.1
245.5 ± 157.7
68.7 ± 20.4
O
O
F
O
O
O
O
O
O
F
25.2 ± 18.9
9.6 ± 3.9
47.3 ± 35.6
6.5 ± 3.5
O
O
O
O
Ac3Az8
O
O
O
O
Ac3Az9
O
2.0 ± 1.2
2.3 ± 1.1
139.3 ± 21.5
53.0 ± 1.5*
O
O
Ac3Az11*
Ac3Az12
6.4 ± 0.2
7.9 ± 6.8
O
O
27.5 ± 19.1
27.1 ± 16.5
155.8 ± 46.8
88.5 ± 27.6
O
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1…………….continued
F1
F2
EC50 (nM) for reversing MDR
BCRP-mediated MRP1-mediated
topotecan resistance DOX resistance paclitaxel resistance
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
P-gp mediated
N
F2
Compounds
N
O
F1
N
O
x y
y
O
x
MCF7-MX100
2008/MRP1
LCC6MDR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
O
Ac5Az1*
Ac5Az2
4 1
4 2
4 2
>57*
>50
66.5 ± 9.2
285.0 ± 77.8
54.0 ± 1.4
57.5 ± 55.9
24.3 ± 7.5
14.8 ± 1.8
32.5 ± 17.7
97.2 ± 11.8*
370.0 ± 41.0
O
O
160.0 ± 84.9
230.0 ± 14.1
320.0 ± 14.1
19.7 ± 0.6
425.0 ± 106.1
136.7 ± 11.7*
217.4 ± 25.0*
225.0 ± 91.9
250.0 ± 30.8*
>1000
O
O
Ac5Az3*
Ac5Az4*
Ac5Az5
>50*
526.0 ± 272.1
746.7 ± 128.6
421.7 ± 75.2
155.7 ± 30.0
O
O
F
4 2 >100*
4 2 >100
4 1 >100*
O
O
O
O
O
O
O
F
Ac5Az7*
64.0 ± 4.2
O
O
O
O
Ac5Az8
Ac5Az9
Ac5Az10
O
4 1 >100
3.6 ± 0.4
4.0 ± 3.3
245.0 ± 35.4
430.0 ± 42.4
O
O
O
O
O
4 2 >100
4 1 >100
2.8 ± 0.9
0.9 ± 0.4
220.0 ± 14.1
>1000
420.0 ± 141.4
605.0 ± 35.4
O
O
O
O
32.5 ± 23.3
23.6 ± 26.3
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Table 1…………….continued
F1
F2
EC50 (nM) for reversing MDR
BCRP-mediated MRP1-mediated
topotecan resistance DOX resistance paclitaxel resistance
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
P-gp mediated
N
F2
Compounds
N
O
F1
N
O
x y
y
O
x
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
MCF7-MX100
2008/MRP1
LCC6MDR
O
Ac5Az11*
Ac5Az12
Ac5Az13
4 1 >100*
28.0 ± 2.8
32.0 ± 5.7
60.0 ± 7.1
135.0 ± 35.4
175.0 ± 17.6*
159.7 ± 35.9
O
O
O
O
4 2
4 0
x
>100
150.8 ± 129.9
223.3 ± 86.2
133.8 ± 54.4
141.0 ± 1.4
O
O
9.2 ± 6.7
348.3 ± 197.5
204.0 ± 33.9
O
Bn
N
F2
N
N
F1
N
O
O
x
O
O
Ac5Az14
Ac5Az15
4
>100
>100
69.5 ± 7.8
38.7 ± 46.9
238.3 ± 102.5
616.7 ± 125.8
678.8 ± 100.5
>1000
O
O
O
O
4
542.5 ± 293.4
371.7 ± 184.7
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1…………….continued
F1
F2
EC50 (nM) for reversing MDR
BCRP-mediated MRP1-mediated
topotecan resistance DOX resistance paclitaxel resistance
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
P-gp mediated
Compounds
N
N
F2
O
F1
N
O
x y
y
O
x
MCF7-MX100
2008/MRP1
LCC6MDR
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Ac12Az1*
Ac12Az2
Ac12Az3
Ac12Az4
Ac12Az5
Ac12Az7
Ac12Az8
Ac12Az9
Ac12Az10
Ac12Az11
Ac12Az12
4 1 >100*
51.5 ± 4.9
47.0 ± 19.8
32.8 ± 24.3
43.5 ± 2.1
35.0 ± 7.1
43.0 ± 4.2
5.2 ± 1.8
24.5 ± 12.0
86.5 ± 16.6*
321.0 ± 43.8
O
O
4 2
4 2
4 2
4 2
4 1
>50
>50
18.0 ± 0.0
45.5 ± 44.5
30.5 ± 20.5
15.5 ± 3.5
17.5 ± 2.1
5.6 ± 2.4
230.0 ± 71.9
171.0 ± 41.9
219.0 ± 40.7
233.3 ± 115.9
172.3 ± 53.7
255.0 ± 63.6
165.0 ± 35.4
650.0 ± 348.7
114.0 ± 15.6
135.0 ± 14.1
360.0 ± 56.6
198.3 ± 7.6
O
O
O
O
F
>100
>100
>100
>100
>100
>100
>100
>100
325.0 ± 7.1
O
O
O
O
O
O
277.5 ± 81.3
264.0 ± 79.2
325.0 ± 63.6
285.0 ± 35.4
650.0 ± 495.0
365.0 ± 91.9
287.5 ± 24.7
O
F
O
O
O
O
O
O
4
1
O
O
O
O
0.9 ± 0.1
1.4 ± 1.0
O
4 2
4 1
4 1
4 2
O
O
20.0 ± 7.1
34.1 ± 32.4
47.0 ± 1.4
34.2 ± 20.8
39.0 ± 39.6
94.0 ± 36.8
O
O
O
O
O
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Table 1…………….continued
F1
F2
EC50 (nM) for reversing MDR
BCRP-mediated MRP1-mediated
topotecan resistance DOX resistance paclitaxel resistance
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
P-gp mediated
Compounds
N
N
F2
O
y
F1
N
O
O
y
O
MCF7-MX100
2008/MRP1
LCC6MDR
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
O
Ac13Az1*
Ac13Az2
Ac13Az3
Ac13Az4
1
2
2
2
>50*
302.5 ± 53.0
302.5 ± 24.7
90.0 ± 7.1
235.0 ± 184.8
235.0 ± 35.5*
960.0 ± 49.0
O
O
22.0 ± 15.6
>100
340.0 ± 172.8
145.0 ± 97.1
264.3 ± 199.0
276.3 ± 120.6
246.7 ± 92.4
243.3 ± 55.1
962.5 ± 47.9
773.3 ± 20.8
966.7 ± 57.7
O
O
O
O
F
>50
230.0 ± 84.9
O
O
O
Ac13Az5*
2
>100
38.0 ± 18.4
24.9 ± 22.6
180.0 ± 19.5*
383.3 ± 32.1
O
O
O
O
O
F
Ac13Az7
Ac13Az8
Ac13Az9
Ac13Az10
Ac13Az11
Ac13Az12
1
1
2
1
1
2
>100
285.0 ± 49.5
4.1 ± 0.6
195.0 ± 102.6
17.7 ± 12.0
2.0 ± 1.3
292.7 ± 65.1
135.0 ± 35.4
180.0 ± 56.6
184.0 ± 33.9
333.3 ± 119.3
272.0 ± 116.0
>1000
O
O
O
O
O
O
>100
436.7 ± 47.3
313.3 ± 49.3
345.0 ± 5.0
>1000
O
O
O
O
>100
1.7 ± 0.8
O
O
O
>100
16.5 ± 3.5
195.0 ± 61.4
145.0 ± 21.2
118.3 ± 61.7
308.0 ± 209.8
59.0 ± 58.0
O
>100
O
O
12.1 ± 4.1
927.5 ± 31.8
O
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1…………….continued
F1
F2
EC50 (nM) for reversing MDR
BCRP-mediated MRP1-mediated
topotecan resistance DOX resistance paclitaxel resistance
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
P-gp mediated
Compounds
F1
O
F2
O
N
O
x y
N
x
N
N
O
N
N
N
y
Bn
MCF7-MX100
2008/MRP1
LCC6MDR
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Ac15(Az1)2
Ac15(Az2)2
1 1
2 2
>100
>100
111.0 ± 12.7
77.5 ± 31.8
205.0 ± 63.6
950.0 ± 70.7
670.0 ± 130.8
O
64.0 ± 31.1
46.0 ± 33.9
25.5 ± 12.0
530.0 ± 169.7
475.0 ± 35.4
579.0 ± 111.7
893.3 ± 110.2
763.3 ± 47.3
560.0 ± 165.2
O
O
O
Ac15(Az3)2
Ac15(Az5)2
Ac15(Az8)2
2 2
2 2
>100
>100
36.0 ± 5.7
O
O
35.5 ± 27.1
O
O
O
O
O
>100
>100
>100
>100
>100
>100
>100
5.3 ± 2.7
3.3 ± 2.1
3.3 ± 4.1
4.3 ± 4.6
457.5 ± 10.6
441.7 ± 14.4
760.0 ± 28.3
750.0 ± 0.0
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
O
1 1
2 2
1 1
2 2
0 0
O
O
O
Ac15(Az9)2
Ac15(Az11)2
Ac15(Az12)2
Ac15(Az13)2
Ac15(Az16)2
Ac15(Az17)2
O
O
O
152.5 ± 123.7
82.5 ± 17.7
242.5 ± 3.5
>1000
278.3 ± 133.6
250.0 ± 70.7
485.0 ± 21.2
>1000
O
O
O
O
O
H
>1000
/
/
O
O
O
1 1
18.5 ± 9.3
10.9 ± 4.7
>1000
O
2
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Table 1…………….continued
F1
F2
EC50 (nM) for reversing MDR
BCRP-mediated MRP1-mediated
topotecan resistance DOX resistance paclitaxel resistance
IC50 towards
L929 (µM)
BCRP-mediated
topotecan resistance
HEK293/R2
P-gp mediated
N
N
F2
Bn
N
Compounds
O
F1
N
O
y
y
O
MCF7-MX100
2008/MRP1
LCC6MDR
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
O
O
Ac16Az1*
Ac16Az2*
Ac16Az3
1
2
2
2
2
1
2
0
>100*
>100*
>100
>100*
>100
>100*
>100
>100
86.1 ± 40.8
36.5 ± 2.1
205.0 ± 7.1
77.7 ± 16.5*
630.0 ± 42.4
O
17.0 ± 1.4
15.8 ± 1.6
98.8 ± 18.3*
137.7 ± 37.0
156.0 ± 26.9*
301.7 ± 161.0
123.0 ± 13.0*
208.0 ± 45.3
670.0 ± 113.1
275.0 ± 35.4
210.0 ± 56.6
333.5 ± 3.5
O
O
54.5 ± 17.1
26.5 ± 6.4
O
O
F
Ac16Az4*
Ac16Az5
19.9 ± 15.1
69.3 ± 47.2
53.5 ± 51.6
58.0 ± 38.2
280.0 ± 141.4
O
O
O
O
O
O
156.3 ± 50.9
48.0 ± 15.9
54.0 ± 5.7
983.3 ± 28.9
285.0 ± 63.6
403.3 ± 55.1
896.7 ± 274.7
O
F
Ac16Az7*
Ac16Az12
Ac16Az13
O
O
O
O
190.0 ± 127.3
O
Ko143
31.4 ± 5.0
>100
8.7 ± 4.9
64.8 ± 28.1
9.0 ± 2.1
63.2 ± 73.6
ND
1950.0 ± 353.6
110.7 ± 42.6
1060.0 ± 207.8
264.0 ± 19.8
32.0 ± 1.4
FD-4e
Cyclosporine A
21.9 ± 9.1
1750.0 ± 150.0
1700.0 ± 141.4
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
A total of 74 compounds were synthesized and their BCRP-, MRP1- and P-gp-modulating activities were determined. EC value was
5
0
used to differentiate the MDR reversal potency of these flavonoid dimers. EC value was presented as mean  standard deviation. N =
50
2-7 independent experiments. L929 is a normal mouse fibroblast cell line. The cytotoxicity of these compounds towards L929 was
tested. Ko143, FD-4e and cyclosporine A are BCRP, MRP1 and P-gp inhibitors, respectively. They were included in the assay as
positive controls *The chemical structure, cytotoxicity, EC values for reversing MRP1-mediated drug resistance have been previously
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
5
0
5
4
reported. Here, we included them in order to make a comparison. ND = not determined. The chemical structures of these compounds
are shown in Supporting Information. / = not applicable.
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Table 2. Comparison of EC of triazole bridged flavonoid dimers for reversing BCRP-, MRP1- and P-gp-mediated drug
5
0
resistance.
BCRP / HEK293/R2 (EC50 nM)
Az1
12
67
7
Az2
Az3
Az4
Az5
Az7
Az8
Az9
Az10
Az11
Az12
Az13
Az14
Az15
Az16
Az17
A
Ac1
Ac2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
52
24
35
14
54
33
90
36
55
38
3
60
25
7
10
4
4
2
3
1
2
3
42
6
57
28
Ac3
Ac5
67
52
303
111
86
285
47
58
44
230
24
15
43
33
20
17
28
32
60
70
543
Ac12
Ac13
Ac15
Ac16
35
5
34
47
303
78
38
285
4
195
153
145
83
36
5
243
190
>1000 19
37
27
156
48
54
Ko143
9
BCRP / MCF7-MX100 (EC50 nM)
Az1
23
Az2
Az3
Az4
Az5
Az7
Az8
Az9
Az10
Az11
Az12
Az13
Az14
Az15
Az16
Az17
B
Ac1
Ac2
30
203
7
185
20
98
4
78
47
20
16
25
26
69
24
7
7
2
1
1
2
4
22
8
177
27
Ac3
18
Ac5
33
160
18
230
46
320
31
64
18
4
24
34
135
39
151
94
223
39
372
Ac12
Ac13
Ac15
Ac16
25
6
235
205
205
340
64
145
46
264
195
18
3
118
308
278
59
250
58
485
280
>1000 11
17
16
20
54
Ko143
9
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 2…………….continued.
MRP1 / 2008/MRP1 (EC50 nM)
Az1
Az2
Az3
Az4
Az5
Az7
Az8
Az9
Az10
Az11
Az12
Az13
Az14
Az15
Az16
Az17
C
Ac1
Ac2
128
151
137
97
170
208
425
230
276
530
99
200
115
137
171
247
475
138
81
207
111
225
233
180
579
302
212
137
245
255
135
458
181
139
298
53
205
156
134
135
272
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Ac3
132
217
219
243
Ac5
250
172
293
220 >1000 175
348
238
617
Ac12
Ac13
Ac15
Ac16
87
165
180
442
650
184
114
333
760
235
950
78
750 >1000
>1000 >1000
156
123
208
670
FD-4e
111
Pgp / LCC6MDR (EC50 nM)
Az1
201
300
155
Az2
Az3
Az4
Az5
Az7
Az8
Az9
Az10
Az11
Az12
Az13
Az14
Az15
Az16
Az17
D
Ac1
Ac2
384
255
350
219
328
161
747
325
967
259
344
422
278
616
481
430
325
414
246
420
285
313
490
69
515
89
Ac3
Ac5
370 >1000 526
156
264
605
650
160
365
141
288
204
679 >1000
Ac12
Ac13
Ac15
Ac16
321
960
670
630
360
962
893
275
198
773
763
210
383 >1000 437
345 >1000 928
>1000 >1000 >1000
403 897
560
983
>1000 >1000
>1000 >1000
334
285
Cyclosporine A
32
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EC ≤10 nM
50
EC = 11 - 60 nM
50
EC = 61 - 150 nM
50
EC =151 - 500 nM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
50
EC > 500 nM
50
The mean EC (nM) value of each flavonoid dimer for reversing BCRP-, MRP1- and P-gp mediated drug resistance was listed. A red
5
0
color gradient was used to quantitatively discriminate from high-to-low MDR reversal potency. The EC values lower than or equal to
5
0
1
0 nM was in red scale, bolded and underlined. N = 2-7 independent experiment. (A) BCRP-modulating activity of HEK293/R2 cell
line, (B) BCRP-modulating activity of MCF7-MX100 cell line, (C) MRP1-modulating activity of 2008/MRP1 cell line and (D) P-gp-
modulating activity of LCC6MDR cell line. The box in grey indicated that the flavonoid dimers have not been synthesized.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
.2.3. MRP1 and P-gp inhibitory activity of flavonoid dimers
The inhibitory activities against MRP1 and P-gp of these 74 compounds were also tested
(Table 1 and Table 2C and 2D). Compounds Ac2Az4, Ac3Az11, Ac5Az1, Ac12Az1, Ac16Az1
and Ac16Az2 displayed the highest MRP1-inhibitory potency with EC less than 100 nM, more
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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2
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6
7
8
9
0
5
0
5
2
potent than the positive control FD-4e with EC =110.7 nM (Table 2C), as previously reported.
5
0
In contrast, all 74 compounds are less potent than the positive control, cyclosporine A (EC =32.0
50
nM), in their P-gp inhibitory activity. Compounds Ac3Az11 and Ac3Az12 showed the highest P-
gp inhibitory potency with EC at 68.7 and 88.5 nM respectively (Table 2D). The linker length
5
0
effect of flavonoid dimers to modulate P-gp and MRP1 transporters has been previously reported
for compounds of general structure I.48-53 A good inhibitor of P-gp or MRP1 should have 4 PEG
linker or 5-6 PEG linker respectively to make the two flavone moieties binding to transporters
selectively.49,52 Shorter or longer linkers would lead to less inhibition towards these two
transporters. For the triazole-bridged flavonoid dimers in the present study, all the potent MRP1
inhibitors have linkers with 13−17 atoms between the two flavonoid moieties, similar in linker
5
4
length to I with n=5-6. For the most active P-gp inhibitors in Table 2D, Ac3Az11 and Ac3Az12
contain 13-16 atoms between the two flavonoids, slightly longer than the optimal length of I with
n=4 (12 atoms in between the two flavonoids). In this context, the 11 compounds, Ac2Az9,
Ac3Az4, Ac3Az9, Ac3Az11, Ac5Az8, Ac5Az9, Ac12Az8, Ac12Az9, Ac13Az9, Ac15(Az8) and
2
Ac15(Az9) , with the most potent activity against BCRP, (Table 1, Table 2A and 2B) contain 13-
2
27 atoms between the two flavonoids. The linker length requirement for inhibitory activity against
BCRP thus appears to be less stringent. This offers an opportunity to design inhibitors which are
more selective for BCRP. Specifically, of the 11 most active compounds, the two bis-triazole
bridged dimers, Ac15(Az8) and Ac15(Az9) have the longest linker length with 21 and 27 atoms
2
2
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Page 29 of 118
Journal of Medicinal Chemistry
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respectively. Indeed, both compounds showed only modest activity towards MRP1 (EC = 442-
5
0
4
58 nM) and essentially no activity towards P-gp (EC >1000 nM) as expected from their longer
50
than the optimal linker length for MRP1 and P-gp inhibition. They are potent (EC = 3-5 nM),
5
0
1
1
1
1
1
1
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5
5
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0
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nontoxic (IC >100 µM towards L929 cells) and selective inhibitors of BCRP.
5
0
2
.2.4. Analogues of Ac15(Az8) and Ac15(Az9) and BCRP selectivity relationship
2 2
We define the selectivity index of an inhibitor for transporter A over transporter B as the
inverse ratio of EC of the inhibitor for transporter A over the EC of the same inhibitor for
5
0
50
transporter B (Table 3). The selectivity index of Ac15(Az8) and Ac15(Az9) is about 86-139 for
2
2
BCRP over MRP1 and >189-303 for BCRP over P-gp depending on the cell lines used. (Table 3).
In order to further improve the BCRP selectivity, several analogues of Ac15(Az8) and
2
Ac15(Az9) have been synthesized by varying the structure of the diacetylene Ac15 as shown in
2
Scheme 4. Their biological data are shown in Table 3. We examine first the effect of replacing
the N-benzyl group in Ac15 with N-phenyl (Ac26) or N-4-pyridinylmethyl (Ac28). Ac26(Az8)2
and Ac26(Az9) are about as potent as the Ac15 counterparts, so both N-benzyl and N-phenyl have
2
significant effect on BCRP-modulation. On the other hand, replacing N-benzyl with N-4-
pyridinylmethyl leads to diminished activity because Ac28(Az8) is much less potent than
2
Ac15(Az8) In fact, the N-group does not appear to be essential at all for BCRP inhibitory activity.
2
.
Ac24(Az8) and Ac25(Az8) are as potent as Ac15(Az8) with CH or O replacing N-benzyl
2
2
2
2
respectively. The difference between Ac24 and Ac22 is that the conformationally flexible three
carbon CH CH CH chain in Ac24 is replaced by an aromatic ring with conformationally rigid
2
2
2
three carbon chain (m-substitution) in Ac22. Interestingly, Ac22(Az8) is slightly more active than
2
Ac24(Az8) . The reason may not be due to the aromatic ring because Ac23(Az8) is less potent
2
2
29
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