Amine linked flavonoid dimers as modulators for P-glycoprotein-based multidrug resistance: Structure-activity relationship and mechanism of modulation

Article abstract of DOI:10.1021/jm201121b

Here we report a great improvement in reversal potency of cancer drug resistance when flavonoid dimers possess a functionally substituted aminopolyethylene glycol linker. The most potent compound, 18, contains a N-benzyl group at the linker. It has many a

Full text of DOI:10.1021/jm201121b

Article
pubs.acs.org/jmc
Amine Linked Flavonoid Dimers as Modulators for
P-Glycoprotein-Based Multidrug Resistance: Structure−Activity
Relationship and Mechanism of Modulation
Kin-Fai Chan,†,‡,∥ Iris L. K. Wong,†,‡,∥ Jason W. Y. Kan,†,‡ Clare S. W. Yan,†,‡ Larry M. C. Chow,*,†,‡
and Tak Hang Chan*,†,‡,§
†Department of Applied Biology and Chemical Technology, State Key Laboratory of Chirosciences, The Hong Kong Polytechnic
University, Hong Kong SAR, China
‡State Key Laboratory for Chinese Medicine and Molecular Pharmacology, The Hong Kong Polytechnic University, Shenzhen, China
§Department of Chemistry, McGill University, Montreal, Quebec, H3A 2K6, Canada
S
* Supporting Information
ABSTRACT: Here we report a great improvement in reversal potency of cancer
drug resistance when flavonoid dimers possess a functionally substituted
aminopolyethylene glycol linker. The most potent compound, 18, contains a
N-benzyl group at the linker. It has many advantages including (1) high potencies
in reversing P-glycoprotein (P-gp) mediated resistance in LCC6MDR cells to
various anticancer drugs with EC50 in the nanomolar range, (2) low toxicity and
high therapeutic index, and (3) preferential inhibition of P-gp over multidrug
resistance protein 1 and breast cancer resistance protein. Compound 18 stimulates
P-gp-ATPase activity by 2.7-fold and mediates a dose-dependent inhibition of
doxorubicin (DOX) transport activity. Lineweaver−Burk and Dixon plots suggest
that 18 is a competitive inhibitor to DOX in binding to P-gp with a Ki of 0.28−
0.34 μM and a Hill coefficient of 1.17. Moreover, the LCC6MDR cell displays
about 2.1-fold lower intracellular accumulation of 18 compared to the wild type,
suggesting that 18 is a P-gp substrate as well.
and more potent agents such as dexverapamil,19 dexniguldipine,20
INTRODUCTION
Treatment of cancer patients with chemotherapeutic drugs
often leads to emergence of tumors with a multidrug resistance
■
valspodar (3),17,21 and biricodar.22,23 The third generation MDR
modulators developed by structure−activity relationships and
combinatorial chemistry approaches included zosuquidar, tariqui-
(MDR) phenotype, which constitutes a major obstacle for
dar, laniquidar, acridonecarboxamide, and the substituted diary-
cancer chemotherapy. Many mechanisms of MDR have been
limidazole derivatives.24,25 Among them, only very few were
described. One of the well-characterized resistance mechanisms
selected for clinical trial and none of them have been approved for
is overexpression of a membrane transporter, permeability
therapeutic use.
glycoprotein (P-gp), which recognizes and pumps a variety of
anticancer drugs out of the cancer cells, resulting in a lowered
Flavonoids are polyphenolic compounds commonly found in
fruits, vegetables, and plant-derived products of the human
intracellular drug accumulation. P-gp belongs to the ATP
diet.26 Humans consume large amounts of flavonoids daily, and
binding cassette (ABC) transporter superfamily, which has a
broad substrate specificity.1,2 Three-dimensional structure of
it is therefore generally accepted that flavonoids are not toxic.
Some flavonoids have been shown to have a low modulating
the mouse homologue of human P-gp revealed that it has a
activity on cancer MDR.27−33 We have previously reported that
synthetic apigenin homodimers, linked by polyethylene glycol
(PEG), can modulate P-gp and multidrug resistance protein 1
(MRP1) in human cancer34−36 and parasitic protozoan
Leishmania.37,38 Such bivalency approach resulted in a signi-
ficantly higher reversal activity in apigenin homodimers than
monomeric apigenin. The most potent apigenin homodimers
for reversing P-gp- and MRP1-mediated drug resistance were 4
and 5 (Chart 1), which have four and five ethylene glycol (EG)
pseudodimeric structure.3 In its nucleotide-free state, the
nucleotide-binding domains (NBDs) were separated by about
30 Å and the two bundles of six transmembrane helices formed
a large internal cavity for substrate binding open to both the
membrane and the inside of the cell.3
There has been intensive search for potent P-gp modulators.
The first P-gp inhibitor was verapamil (1) developed by Tsuruo
et al.4 Subsequently, a range of compounds including calcium
channel blockers,5,6 calmodulin inhibitors,7,8 indole alkaloids,9,10
cyclosporine A (2),11−14 quinolines,15 and steroids16−18 have been
demonstrated to have P-gp inhibitory activity to different extents.
The second generation MDR modulators resulted in the less toxic
Received: August 22, 2011
Published: February 9, 2012
© 2012 American Chemical Society
1999
dx.doi.org/10.1021/jm201121b | J. Med. Chem. 2012, 55, 1999−2014

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Article
a
Chart 1. Chemical Structures of Compounds 1−10
Scheme 1. Synthesis of Flavonoid Dimers 13 and 14
a
Reagents and conditions: (a) K2CO3, DMF, reflux, 2 h; (b) trifluoroacetic
acid, CH2Cl2, 0 °C to room temp, 2 h.
a
Scheme 2. Synthesis of Compounds 15−37
units, respectively.34,36 Modulating activity of flavonoid dimers
has been further optimized by structural modification of the A
ring.35,36 In general, replacement of the OH group in the A ring
with H (e.g., 6) led to more potent P-gp modulators.
Furthermore, nonpolar and hydrophobic substituents (e.g.,
methyl, ethyl, or fluoro groups) were better than polar and
hydrophilic substituents (e.g., hydroxyl groups) at the C3, C6,
and C7 positions.35,36 The EC50 of compound 7 (Chart 1) for
vincristine was determined to be 110 nM. This efficacy was
close to other highly efficient modulators such as tariquidar
(EC50 = 22−38 nM for vincristine).
These flavonoid dimers are, however, highly hydrophobic
and barely soluble in water. Attempts to use these compounds
for in vivo animal experiments proved to be impractical
(data not shown). In order to improve their physicochemical
properties as potential drug candidates, we must introduce
some hydrophilic groups into the structure. Since we already
knew that introduction of hydrophilic groups into the flavonoid
moiety reduced potency, it leaves the linker as a possible site for
structural modification. Here we synthesize a series of new
flavonoid dimers with nitrogen-containing PEG linker and
evaluate their P-gp-modulating activity, cytotoxicity against
cancer cells and normal cells, and their specificity toward
MRP1 and breast cancer resistance protein (BCRP). The most
effective amine linked flavonoid dimer has been further investi-
gated regarding its mechanism of reversing P-gp-mediated drug
resistance.
a
Reagents and conditions: (a) for compounds 15−30, K2CO3, RX,
ACN, or DMF, reflux, 3 h; (b) for compounds 31 and 32, RCOCl,
pyridine, 0 °C to room temp; (c) for compound 33, succinic
anhydrite, pyridine, room temp, 4 h; (d) for compound 34, coupling
with t-Boc-alanine followed by acid hydrolysis; (e) for compounds
35−37, RSO2Cl, pyridine, 0 °C to room temp.
corresponding 4′-hydroxyflavones 11a−f, prepared as previ-
ously reported35 in dimethylformamide (DMF) under basic
condition at refluxing temperature, the desired N-Boc protected
flavonoid dimers 13a−f with various substituents on the flavone
rings were obtained in reasonable yield. Their dimeric nature
was evident from the high-resolution mass spectrum. Treat-
ment of these compounds with trifluoroacetic acid (TFA) in
CH2Cl2 followed by extraction at basic pH led to the
corresponding flavonoid dimers 14a−f with the N−H group
at the linker region in high yield. This synthetic pathway offers
advantages and flexibility to access desired flavonoid dimers for
structure−activity relationship (SAR) study at the linker region.
RESULTS AND DISCUSSION
■
I. Chemistry. By use of 6 as the starting template, the
synthesis of amine-containing PEG linker is considerably
simplified by replacing the central oxygen with nitrogen. The
designed flavonoid dimers were synthesized as summarized in
Schemes 1 and 2. As depicted in Scheme 1, by reaction of the
tert-butyloxycarbonyl (N-Boc) protected ditosylate 12 with
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Journal of Medicinal Chemistry
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It should be mentioned that the hydrochloride salt of
compound 14a was obtained simply by treatment with
concentrated hydrochloric acid followed by evaporation to
dryness.
benzyloxy group. Addition of a fluorine atom at the C6 position
(13f) of the A ring resulted in a moderate efficacy with a RF of
31.9 at 1.0 μM. In terms of cytotoxicity, the unsubstituted com-
pound (13a) and C5-methoxy substituted compound (13d)
displayed killing activity to the cancer lines (IC50 < 8.0 μM) and
normal cell L929 (IC50 < 11.1 μM). In contrast, C6-methyl sub-
stituted compound (13c) showed no cytotoxicity to all of the
different cell lines (IC50 > 100 μM).
After preliminary biological screening of these flavonoid
dimers, 14a−f and their hydrochloride salts 14a and 14f
showed not only good P-gp modulating activity but also greatly
improved aqueous solubility compared with the PEG linked
flavonoid dimer 6 or 7. These encouraging results prompted us
to carry out further structural optimization at the amino linker
region. Compound 14a was selected to be the focus of atten-
tion because of inexpensive starting material 2-hydroxyaceto-
phone, which is commercially available in bulk. Treatment of
14a with various alkyl halides in acetonitrile (ACN) or DMF at
refluxing temperature afforded tertiary amine derivatives (15−
30) in good yield (Scheme 2). Amide derivatives (31, 32) were
also obtained under similar conditions in high yield by mixing
14a with the corresponding acid chlorides. Amide derivative
33 was furnished by treatment of 14a with succinic anhydride
in pyridine at room temperature. Amide derivative 34 was
prepared in two steps from commercially available Boc-Ala-OH
followed by acid hydrolysis using TFA. Treatment of 14a with
various sulfonyl chlorides in pyridine at 0 °C afforded
sulfonamide derivatives (35−37) in reasonably good yields.
II.A. Structure−Activity Relationship Study. Effect of
Amine Linked Flavonoid Dimers on Reversing Paclitaxel
Resistance in MDA435/LCC6MDR Cells. We employed two cell
lines in this study, namely, MDA435/LCC6 (an estrogen-
independent human breast cancer cell line) and its P-gp-
transfected derivative (MDA435/LCC6MDR).39 LCC6MDR
(IC50 for paclitaxel of 153.1 2.9 nM) displayed about 95.7-
fold greater resistance than LCC6 cells (IC50 of 1.6 0.3 nM)
(Table 1). Cytotoxicity of flavonoid dimers alone, measured by
the IC50 toward LCC6, LCC6MDR, and a normal mouse
fibroblast cell line L929, ranged from 2.4 to above 100 μM. At
the concentration we used for assaying P-gp reversing activity
(0.5 or 1.0 μM), there was at least 80% survival (data not
shown). Flavonoid dimers synthesized can be structurally
divided into six series (A−F) according to the chemical sub-
stitutions on the amine linker. Table 1 summarizes the results
of cytotoxicity and P-gp-modulating activity of all flavonoid
dimers.
II.A.1. Boc-Group Protected Amine Linked Flavonoid
Homodimers (Series A, Table 1). All flavonoid dimers in this
series have a Boc group protecting the amine group on the PEG
linker. The unsubstituted parent compound 13a displayed
a promising P-gp-modulating activity with a RF of 69.6 at
1.0 μM. The N-Boc group in the linker offers a significant
improvement over 635 (RF = 17.2), which has a central oxygen
in the PEG linker. Compound 13a was about 20-fold more
potent than 1 (RF = 3.5). Addition of a methoxy group at the
C3′ position of the B ring (13b), however, caused a reduction
in reversal potency with a RF of 16.8 at 1.0 μM, suggesting that
hydrophobicity at position 3′ in the B ring is unfavorable for P-
gp-modulating activity. On the other hand, addition of a methyl
group at the C6 position (13c) or a methoxy group at the C5
position (13d) of the A ring displayed an improved efficacy
with a RF of 139.2 and 109.4 at 1.0 μM, respectively. This
result suggests that the increased hydrophobicity in ring A is
important to produce efficient P-gp inhibitor. However, a bulky
benzyloxy group at the C3 position of the C ring (13e) showed
no modulating activity with a RF of 1.2 at 1.0 μM. This loss of
P-gp inhibitory activity may be due to the steric hindrance of
II.A.2. N−H Linked Flavonoid Homodimers (Series B,
Table 1). All N−H linked flavonoid dimers in this series have
their Boc group removed from the linker by acid hydrolysis.
Similar to their Boc-protected parent compounds of 13a (RF =
69.6) and 13c (RF =139.2), the unsubstituted 14a and C6-
methyl substituted 14c, both displayed potent reversal activity
with RF of 139.2 at 1.0 μM. Interestingly, C3′-methoxy sub-
stituted compound 14b (RF = 1.1) and C5-methoxy sub-
stituted compound 14d (RF = 1.2) completely lost their P-gp-
modulating potency compared to corresponding compounds
13b (RF = 16.8) and 13d (RF = 109.4). In contrast, C3-
benzyloxy substituted compound 14e (RF = 127.6) regained
the promising reversal potency compared to the corresponding
compound 13e (RF = 1.2). These data suggest that there may
be some positive or negative interacting activity between the
Boc group on the linker and the substitution on the flavone
moiety and their combined effect would influence the P-gp-
mediated resistance reversal potency. Nevertheless, all N−H
linked dimers in this series were found to exhibit toxicity
toward all cell lines tested (IC50 < 23.8 μM), suggesting that a
free N−H group on the linker would potentiate its cytotoxicity.
II.A.3. Functional-Group-Substituted Amine Linked Fla-
vonoid Homodimers (Series C−F, Table 1). The amine group
on the linker in series C−F was substituted with various fun-
ctional groups such as alkyl group (C), amide group (D),
sulfonamide group (E), and benzyl group or pyridylmethyl
group (F). We found that the P-gp inhibitory potency and
toxicity toward L929 varied greatly among these amine linked
flavonoid dimers. In general, all compounds exhibited moderate
to potent paclitaxel reversing activity at 1.0 μM (RF = 18.9 to
139.2), except those that showed no reversal potency included
amides 33 and 34 and sulfonamide 37.
To further differentiate reversal potency of those active
compounds, we tested them at lower concentration (0.5 μM).
The presence of a benzyl group on 18 (RF = 58.9) conferred
the highest activity. In addition, reversal potency has been
diminished slightly by unfavorable electron-withdrawing
substituents on the phenyl ring, such as fluoro (25, 27, and
28), difluoro (29), trifluoro (30), trifluoromethyl (26), nitro
(19), and methyl ester (20). The distance between the nitrogen
atom and the phenyl ring appeared to be a crucial element, as
the lengthened compound 23 or 24 was less potent than 18.
One methylene group seems to be optimal. Besides, a phenyl
ring without any nitrogen substitution seems to be favorable, as
a pyridyl ring (21, 22) also displayed much lower modulating
activity than 18. Moreover, compounds 31, 32, 35, and 36 were
prepared to explore the effects of replacing the benzylic carbon
with other functional groups, such as carbonyl and sulfonyl. It
was found that their modulating activities were much lower
than that of 18. This may be attributed to the fact that both
amide and sulfonamide bonds are less basic than amines.
Finally, we synthesized compounds 15, 16, and 17 to
investigate the role of the phenyl ring of 18. These derivatives
generally displayed much lower modulating activity than 18. It
therefore seems reasonable to hypothesize that there may be an
2001
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Journal of Medicinal Chemistry
Article
a
Table 1. Cytotoxicity and Paclitaxel-Resistance Reversal Activity of Amine Linked Flavonoid Dimers
f
f
cytotoxicity IC50 (μM)
IC50 of paclitaxel of LCC6MDR [nM] (RF)
compd at 1.0 μM compd at 0.5 μM
2.2 0.5 13.8 3.7
b
compd
13a
series
LCC6MDR
LCC6
L929
A
A
A
A
A
A
B
B
B
B
B
B
C
C
C
C
C
D
D
D
D
E
E
E
F
6.2 0.8
6.9 2.9
>100
8.0 2.5
8.3 1.4
>100
11.1 0.0
33.3
(69.6)
(16.8)
(139.2)
(109.4)
(1.2)
(11.1)
(8.7)
(9.6)
(14.9)
(1.1)
(11.3)
(72.9)
(0.8)
(13.9)
(1.2)
(54.7)
(72.9)
(26.9)
(5.0)
(5.3)
(9.6)
(12.5)
(18.9)
(7.5)
(1.1)
(1.0)
(12.8)
(5.8)
(1.4)
(58.9)
(43.7)
(2.1)
(6.9)
(15.8)
(31.2)
(37.3)
(43.7)
(36.5)
(49.3)
(3.3)
(6.2)
(2.0)
nd
13b
13c
13d
13e
13f
14a
14b
14c
14d
14e
14f
15
9.1 3.6
1.1 0.3
1.4 0.3
131.4 4.9
4.8 1.4
1.1 0.1
134.9
17.6 1.8
16.0 3.1
10.3 3.2
137.6 20.6
13.5 0.5
2.1 0.3
194.0 5.1
11.0 2.0
124.9 10.4
2.8 0.6
2.1 0.4
5.7 0.2
30.6 3.8
28.9 9.5
16.0 0.8
12.2 8.5
8.1 1.4
20.4 2.0
144.6
>100
5.4 0.5
>100
4.6 0.2
>100
5.5 0.4
nd
>100
>100
>100
(31.9)
(139.2)
(1.1)
2.4 0.7
23.8 12.8
7.0 1.4
10.2 3.8
4.4 0.1
3.3 0.5
17.3 3.8
14.8 5.9
17.7 3.7
4.9 0.1
>100
3.3 1.0
12.8 1.1
7.5 0.9
8.8 2.7
4.3 0.5
5.3 1.0
10.3 2.2
10.7 3.3
13.5 0.5
8.6 3.4
>100
5.9 0.6
nd
6.0 0.6
nd
1.1 0.1
126.6 11.4
1.2 0.1
1.4 0.1
1.6 0.3
4.7 0.6
3.5 0.5
1.3 0.0
2.7 1.7
2.1 0.5
3.6 0.7
164.5
(139.2)
(1.2)
4.2
(127.6)
(109.4)
(95.7)
(32.6)
(43.7)
(117.8)
(56.7)
(72.9)
(42.5)
(0.9)
5.7 0.4
6.9 2.0
6.9 1.4
18.1 3.4
17.0 4.3
9.5 0.6
>100
16
17
23
24
31
>100
>100
32
>100
>100
95.0
33
>100
78.4 8.1
4.4 0.0
27.5 8.4
>100
nd
34
4.6 0.2
42.6 5.4
>100
nd
173.1 40.0
7.6
(0.9)
145.9 13.8
12.0
35
13.4 3.9
>100
(20.1)
(21.6)
(1.3)
36
7.1 0.9
116.8 9.5
1.1 0.1
1.2 0.1
8.1 3.5
5.5 1.4
1.6 0.1
1.4 0.1
1.7 0.4
1.5 0.2
1.3 0.0
1.4 0.0
1.9 0.3
8.9 2.6
43.9 5.2
1.8 0.3
2.0 0.2
148.0 13.9
26.3 5.6
111.2 10.2
2.6 0.6
3.5 1.2
72.3 15.4
22.3 0.2
9.7 0.1
4.9 1.8
4.1 1.1
3.5 0.5
4.2 1.0
3.1 0.6
46.8 23.2
24.7 4.9
76.4 10.4
nd
37
>100
>100
nd
18
>63
>75
85.0 5.0
88.0 6.1
11.4 4.4
26.4 2.7
10.0 2.8
19.7 0.1
32.7 8.7
>100
(139.2)
(127.6)
(18.9)
(27.8)
(95.7)
(109.4)
(90.1)
(102.1)
(117.8)
(109.4)
(80.6)
(17.2)
(3.5)
19
F
>90
86.8 1.8
33.3 10.8
>100
20
F
64.8 14.1
>100
21
F
22
F
29.7 16.4
>83
10.0 1.2
>100
25
F
26
F
>100
>100
27
F
>100
>100
28
F
>100
>100
>87
29
F
33.6 4.3
>100
32.5 11.0
>75
42.2 2.1
>100
30
F
6
nd
nd
nd
1
63.9 1.7
14.6 2.2
2.8 0.6
nd
63.8 0.1
25.3 4.3
8.3 1.5
nd
89.2 8.2
>100
3
(85.1)
(76.6)
(1.0)
2
33.9 5.2
nd
nd
nd
c
d
DMSO
138.4 8.6
(1.1)
e
LCC6MDR
LCC6
nd
nd
nd
153.1 2.9
(1.0)
nd
nd
e
nd
nd
nd
1.6 0.3
(95.7)
a
IC50 value toward paclitaxel was determined in the presence of 1.0 or 0.5 μM modulator. Relative fold (RF) represents fold-change in drug
sensitivity. N = 1−4 independent experiments, and the values were presented as the mean standard error of mean. RF = (IC50 without
modulator)/(IC50 with 1.0 or 0.5 μM modulator). Known P-gp inhibitors 1, 2, 3, and 6 were included for comparison. All compounds were
b
c
d
dissolved in DMSO for testing. 0.1% of DMSO was used as solvent control for testing the P-gp modulating activity. 0.05% of DMSO was used as
e
solvent control for testing the P-gp modulating activity. The cytotoxicity of LCC6 and LCC6MDR to paclitaxel was tested in the absence of
modulators. For cytotoxicity assay, IC50 values of different flavonoid dimers for LCC6, LCC6MDR, and L929 cell lines were determined. N = 1−3
f
independent experiments, and the values were presented as the mean standard error of mean. L929: mouse fibroblasts. nd: not determined.
interaction between the phenyl ring of 18 and the amino acid
with aromatic substituents of P-gp such as tryptophan, tyrosine,
and phenylalanine through π−π interaction.
Other than modulating activity, the amine linker also plays an
important role in determining its cytotoxic effect. For a proper
comparison, three levels of toxicity were employed: high
(cytotoxicity, IC50 < 20 μM), moderate (20−60 μM), and low
(cytotoxicity, >60 μM). From series B to F, all N−H linked
homodimers (14a−f) (series B) displayed a high level of
toxicity toward cancer cells and normal fibroblast. In series C,
all compounds (15−17, 23) were at high levels of toxicity to
cancer cells and L929 cells except 24 which was nontoxic to
both cancer cells. In general, flavonoid homodimer possessing
unsubstituted N−H or small N-alkyl linkers would result in
high killing activity toward cancer cells or mouse fibroblast.
When the bulkiness of the amine linker is increased, the
cytotoxicity level would generally be reduced. From the series
D, E, and F, 3 out of 18 dimers (20, 22, and 34) displayed a
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Journal of Medicinal Chemistry
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a
Table 2. EC50 and Therapeutic Indexes of Amine Linked Flavonoid Dimers for Lowering Anticancer Drug Resistance
EC50 for reversing anticancer drug resistance (nM)
compd
paclitaxel
vinblasinte
vincristine
DOX
nd
daunorubicin
mitoxantrone
therapeutic index
13a
14a
15
18
19
24
25
26
27
28
1
320 40
305 35
200
nd
nd
nd
nd
34.7
nd
nd
nd
nd
nd
19.3
nd
nd
nd
nd
nd
34.5
148 18
173 27
179 32
131 13
95 25
nd
90 20
nd
574.3
465.6
38.0
189
250
255
150
1
5
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
77.3
nd
nd
nd
nd
nd
218.0
>558.7
>337.2
208.4
179 44
258 103
428 40
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
503 92
385 35
245 23
nd
nd
a
EC50 values were presented as the mean standard error of mean. N = 1−7 independent experiments. Therapeutic index = (IC50 of modulators
towards L929 fibroblasts)/(EC50 of modulators for reversing paclitaxel resistance in LCC6MDR cells). nd: not determined.
a
Table 3. Effect of 18 on P-gp, MRP1, and BCRP Modulating Activity
IC50 [nM] (RF)
b
cell line
LCC6MDR
overexpress
resistant to
no modulator
18
+ve control
P-gp
nd
paclitaxel
nd
153.1 2.9 (1.0)
1.6 0.3 (95.7)
404.2 41.9 (1.0)
50.3 5.6 (8.0)
563.4 41.1 (1.0)
22.4 2.4 (25.2)
2.6 0.6 (58.9)
1.8 0.5 (85.1)
44.8 10.4 (9.0)
40.5 8.3 (13.9)
LCC6
2008/MRP1
2008/P
MRP1
nd
DOX
nd
192.0 58.0 (2.1)
147.0 3.9 (3.8)
HEK292/R2
HEK293/pcDNA3.1
BCRP
nd
topotecan
nd
a
Modulating activity of 18 and +ve control modulators (all at 0.5 μM) on P-gp, MRP1, and BCRP were investigated using LCC6MDR, 2008/
MRP1, and HEK293/R2 cells, respectively (N = 2−4 independent experiments, and the values are presented as the mean standard error of mean).
IC50 values toward paclitaxel, DOX, and topotecan in these three cell lines were determined with or without modulators to determine RF. IC50 values
b
were also determined for their parental cell lines (LCC6, 2008/P, and HEK293/pcDNA3.1) for reference. +ve controls used for modulating P-gp,
MRP1, and BCRP (all at 0.5 μM) are compounds 2, 8, and 9, respectively. nd: not determined.
high level of toxicity toward normal cells L929, as their IC50
values were below 11 μM. A total of 10 compounds (18, 19, 27,
28, and 30 in series F; 31, 32, and 33 in series D; and 36 and
37 in series E) were found to be nontoxic to the cancer cells
and L929 cells. Overall, a benzyl group appeared to be a
desirable substitution for making a potent P-gp inhibitor while
maintaining low toxicity to the normal cells.
II.B. Effect of Compound 18 on Reversing P-gp toward
Other Anticancer Drugs. Among the most potent N-linked
flavonoid dimers listed in Table 1, we have determined the
EC50 values for modulating P-gp toward various anticancer
drugs from a selected group of compounds (13a, 14a, 15, 18,
19, and 24−28). EC50 refers to the concentration of modulator
required to reduce the IC50 by half compared to control
without modulators. Compounds 18 and 26 were the most
active N-linked flavonoid dimers in modulating P-gp toward
of >558.7), and 28 (therapeutic index of >337.2). These com-
pounds are considerably less toxic than 1 (therapeutic index
of 208.4).
On the basis o fthe P-gp-modulating activity and cytotoxicity
of the amine linked flavonoid dimers, we can draw the
following conclusion related to their structure−activity relation-
ships: (1) introduction of Boc group on the amine linker would
greatly improve the P-gp-modulating activity compared to the
previously reported PEG linked flavonoid dimers (e.g., 6); (2)
introduction of a hydrophobic group at the C6 or C5 position
of ring A has a dramatic effect on the P-gp-mediated MDR
reversal potency, and its effect is dependent on the size of the
group; (3) removal of a Boc group from the amine linker would
markedly potentiate its toxicity in a number of compounds,
suggesting that flavonoid dimers with free N−H linker
would be a toxic P-gp chemosensitizer; (4) the nature of the
N-substituent on the amine linker has a strong influence on both
P-gp-modulating activity and cytotoxicity. Balancing all the above
factors, we have chosen 18 for further investigation.
II.C. Preferential Inhibition P-gp over MRP1 and BCRP by 18.
Other than P-gp, MRP1 and BCRP have also been implicated
in clinical MDR. Here, we investigated whether 18 might have
any MRP1- and/or BCRP-modulating activity. At 0.5 μM, 18
restored chemosensitivity of LCC6MDR to paclitaxel, with
RF = 58.9 (Table 3). At the same concentration (0.5 μM), 18
mediated a weak modulating activity for both MRP1 (RF = 2.1
in 2008/MRP1 cells) and BCRP (RF = 3.8 in HEK293/
R2 cells). Positive controls 2, 8, and 9 can effectively modulate
P-gp, MRP1, and BCRP with RF equal to 85.1, 9.0, and 13.9,
paclitaxel with EC50 equal to 148
18 nM and 150 nM,
respectively (Table 2). Compound 18 was also very potent in
modulating P-gp-mediated resistance toward vinblastine,
vincristine, DOX, daunorubicin, and mitoxantrone with EC50
in nanomolar range between 90 and 179 nM (Table 2). These
EC50 values are lower than that of 1 and is within 2- to 3-fold
difference from a highly effective modulator like tariquidar
(EC50 = 60 nM for paclitaxel resistance).40
We have also measured the therapeutic index (ratio of
IC50 toward L929 to EC50 for reversing paclitaxel resistance)
for compounds listed in Table 2. The most nontoxic com-
pounds (highest therapeutic index) are 18 (therapeutic index of
574.3), 19 (therapeutic index of 465.6), 27 (therapeutic index
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respectively. These data suggest that compound 18 is more
likely a preferential inhibitor toward P-gp than MRP1 or BCRP.
III. Mechanism of P-gp Modulation by 18. How does
compound 18 modulate P-gp? It has been suggested that 10
binds at the NBD of P-gp, with C4 carbonyl and C7 phenolic
functionalities of apigenin mimicking N1 and N7 of ATP.27,28
Interestingly, we previously observed that apigenin dimer 4 can
stimulate, instead of inhibit, ATPase activity of P-gp. This is
more in line with the general observation that P-gp substrates
can stimulate its ATPase activity. On the contrary, compounds
that bind to the NBD of P-gp would inhibit ATP hydrolysis.34
This suggested that 4 is not binding to the NBD region of P-
gp.34 Others have suggested that alkyl-substituted flavonoids
may bind to the hydrophobic steroidal interacting region or the
drug binding sites of the transmembrane domain (TMD) of P-
gp.30 Our previous observation that hydrophobic substitution
in the flavonoid moiety can enhance modulating activity of
flavonoid dimers was consistent with the latter model.35 Here,
we investigated if 18, now carrying a hydrophilic amine linker,
will still be able to bind to the drug binding site of P-gp.
III.A. Effect of 18 on P-gp-ATPase Activity. We first
examined the effect of 18 on P-gp-ATPase activity. Compound
1, being an inhibitor as well as a substrate of P-gp, is one of
the best simulators of P-gp-ATPase. At 200 μM, 1 strongly
increased the P-gp-ATPase activity over the basal level by 5.3-
fold (Figure 1). Amine linked flavonoid dimer 18, at 200 μM,
Figure 2. Effect of 1 and 18 on intracellular DOX accumulation in
LCC6MDR cells. LCC6MDR cells were incubated with 20 μM DOX
for 150 min at 37 °C with different concentrations of 18 (0, 0.01, 0.05,
0.1, 0.5, 1, and 3 μM) and 1 (0, 0.01, 0.05, 0.1, 0.5, 1, 3, and 8 μM).
0.4% of DMSO was used as negative control. After the incubation
period, cells were lysed and the supernatant was measured for the
DOX level by spectrofluorometry. N = 2−5 independent experiments.
The results are presented as the mean
standard error of mean:
(∗) P < 0.005, (∗∗) P < 0.001, and (∗∗∗) P < 0.0005 relative to the
negative control (LCC6MDR).
cells in a dose-dependent manner (Figure 2). Minimum
concentration required for 18 and 1 to restore DOX
accumulation of LCC6MDR to that of LCC6’s level is about
0.5 and 8 μM, respectively (Figure 2). Compound 18 is there-
fore roughly 16-fold more potent than 1 in inhibiting the DOX
transport activity of P-gp.
III.C. Compound 18 Is a Competitive Inhibitor of DOX in
Binding to P-gp. Lineweaver−Burk and Dixon plots were
employed to further characterize the inhibition mechanism of
18 on P-gp’s DOX transport activity. Lineweaver−Burk analysis
found that Vmax was unaffected, whereas Km was decreased in
the presence of either 1 or 18, suggesting that 1 and 18 were
both competitive inhibitors of DOX in binding to P-gp (Figure 3A
and Figure 3B). From a plotting of the slopes of the
Lineweaver−Burk plot versus the corresponding modulator
concentrations used, Ki can be determined for 1 (Ki = 1.75 μM,
inset of Figure 3A) and 18 (Ki = 0.34 μM, inset of Figure 3B).
The Dixon plot was also used to determine Ki for 1 (1.75 μM,
Figure 3C) and 18 (0.28 μM, Figure 3D). These values are very
similar to the Ki values determined using the Lineweaver−Burk
plot. The linear regression line obtained from plotting the
slopes of the Dixon plot versus reciprocal of substrate con-
centration can be used to distinguish a competitive, noncom-
petitive, or partially competitive relationship between inhibitor
and substrate.41,42 A competitive inhibitor will fit a line through
the origin, whereas a noncompetitive inhibitor would not.41,42
It was found that the regression lines almost coincide with the
origin for 1 (inset of Figure 3C) and 18 (inset of Figure 3D),
suggesting that both 1 and 18 are competitive inhibitors of DOX
binding to P-gp, which is consistent with the Lineweaver−Burk
analysis.
Figure 1. Effect of 1 and 18 on P-gp-ATPase activity. Sodium
vanadate inhibitable ATPase (P-gp ATPase) was studied as described
in Experimental Section. P-gp ATPase was measured in the absence
(basal activity) or presence of P-gp modulators (1 or 18, both at
200 μM). The latter is normalized to the basal activity and presented
as stimulation fold (SF). Results are presented as the mean standard
error of mean. N = 2 independent experiments.
can also stimulate P-gp-ATPase over the basal level by 2.7-fold
(Figure 1). This result suggests that 18, even though with the
more hydrophilic amine group, is similar to 4 in not binding to
the NBD region of P-gp.34 Compound 18, like 1, may bind to
the substrate recognition site of P-gp and stimulate its P-gp-
ATPase activity.
III.B. 18 Can Increase Intracellular DOX Accumulation in
MDA435/LCC6MDR Cells. We investigated whether the modu-
lation of P-gp-mediated drug resistance by 18 is associated with
a concomitant increase in DOX accumulation. In the absence of
modulator, DOX accumulation in parental LCC6 cells was
about 2.9-fold (P < 0.001) higher than that of LCC6MDR cells
(Figure 2). This is probably due to P-gp-mediated DOX efflux.
Both 18 and 1 can increase DOX accumulation in LCC6MDR
The Ki of 18 determined by both Lineweaver−Burk and
Dixon plots was 0.28−0.34 μM. This is about 5.1- to 6.3-fold
lower than the average Ki of 1 (1.75 μM) determined from
Lineweaver−Burk and Dixon plots, indicating that 18 is more
effective than 1 in displacing the DOX from P-gp. This is
consistent with our observation that 16-fold more of 1,
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Figure 3. Lineweaver−Burk and Dixon analyses of the relationship between DOX and 1 or 18 in binding to P-gp. LCC6MDR cells were incubated
with different concentrations of DOX (1.5, 3.0, 4.4, 13.3, 20.0, 30.0, and 40.0 μM) in the presence of 1 (0. 0.06, 0.25, 0.5, 0.75, or 1.0 μM) or 18
(0, 0.006, 0.025, 0.05, 0.075, or 0.1 μM). After incubation, the intracellular DOX level was measured by cell lysis followed by fluorescence
spectrophotometry. Reciprocal of DOX retention rate is plotted against reciprocal of DOX concentration used (Lineweaver−Burk plot) in the
presence of 1 (A) or 18 (B). Apparent Ki is determined by linear regression analysis of the slopes obtained from the double reciprocal plots versus
the concentration of 1 or 18 used (insets of (A) and (B)). The relationship is also analyzed by Dixon plot: (C) 1 and (D) 18. The reciprocal of
DOX retention rate was plotted against concentration of 1 or 18. The intersected point of the lines represented the Ki. The slope of the Dixon plot
was plotted against reciprocal of DOX concentration to determine the relationship. N = 3−8 independent experiments, and the values were
presented as the mean standard error of mean.
Figure 4. Hill coefficients of 1 and 18 for the DOX accumulation assay. LCC6MDR cells were incubated with 20 μM DOX in the presence of
different concentrations of 1 (A) or 18 (B) (40, 30, 20, 10, 8, 6, 4, 2, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 μM). After incubation,
the intracellular DOX level was measured by cell lysis followed by fluorescence spectrophotometry. The value of the Hill coefficient was determined
by nonlinear regression of the following equation: Y = bottom + (top − bottom)/1 + 10(logEC −X)Hill coefficient, where “bottom” is the baseline and “top”
50
is the maximum effect, EC50 is the dose giving half the maximum effect, X is the modulator concentration (log). The Hill coefficient was presented as
the mean standard error of mean from four to five independent experiments.
compared with 18, is needed to fully restore DOX
accumulation in LCC6MDR cells to the parental LCC6’s
level (Figure 2). In addition, the Ki of 18 (0.28−0.34 μM) for
inhibiting P-gp-mediated DOX efflux is similar to the EC50 of 18
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(131 nM) for chemosensitizing LCC6MDR to DOX (Table 2).
This result suggests that the chemosensitization mechanism of 18
is by virtue of its direct inhibition of P-gp-mediated DOX efflux
(Figure 3), thereby increasing the intracellular DOX retention
(Figure 2) and finally restoring DOX sensitivity (Tables 1 and 2).
In order to further understand the interaction between P-gp and
18, the Hill slope (Hill coefficient) was determined. A Hill co-
efficient can reveal the biophysical nature of the receptor−ligand
interaction.43 A Hill coefficient of ∼1 implies a single binding site
with a simple mass action displacement. A Hill coefficient of >1 is
indicative of cooperative binding, and a Hill value of <1 suggests
negative cooperativity.43 The Hill coefficient has been used to study
the interaction between modulators and P-gp with respect to its
drug efflux or ATPase activity.44−46 Here, displacement of DOX
from P-gp by 18 or 1 revealed a Hill coefficient of 1.17 0.04 and
0.93 0.06, respectively (Figure 4). This suggests that there is non-
cooperative transport of DOX in the presence of 18 or 1. In other
words, there is independent binding of 18 or 1 with DOX.
In view of the stimulatory P-gp-ATPase activity and its
competitive inhibition on DOX’s transport, there is the
possibility that compound 18 is itself a P-gp substrate as well.
In order to further investigate whether compound 18 can be
transported by P-gp, an intracellular accumulation assay of 18
was carried out. The LCC6MDR cell line was found to accu-
mulate less 18 than the LCC6 cell line by 1.7-fold and 2.1-fold
after incubating with 10 and 100 μM 18, respectively (Figure 5).
This indicates that compound 18 is a P-gp substrate.
flavonoid dimer is compound 18 containing a benzyl group at
the amine functional group. Compound 18 significantly
resensitizes LCC6MDR cells to various anticancer drugs
including paclitaxel, vinblastine, vincristine, DOX, daunorubi-
cin, and mitoxantrone with nanomolar EC50 values (EC50
ranged from 90 to 179 nM). Its mechanism of MDR
modulation is associated with an increase in intracellular drug
accumulation induced by directly inhibiting transport activity of
P-gp. Kinetic characterization suggests that 18 likely acts as a
competitive inhibitor of DOX. P-gp-ATPase activity indicates
that 18, like 1, can stimulate P-gp-ATPase activity by binding to
the substrate recognition site. The significant advantage of 18
includes its noncytotoxicity (IC50 for L929 of 85.0 μM) and its
high therapeutic index (therapeutic index of 574.3). Because of
the amine functional group, the hydrochloride salt of 18 can be
readily prepared to improve water solubility. Pharmacokinetic
studies and in vivo animal experiments with 18 will be reported
in due course.
EXPERIMENTAL SECTION
■
General. All NMR spectra were recorded on a Bruker MHz
DPX400 spectrometer at 400.13 MHz for 1H and 100.62 MHz for 13C.
All NMR measurements were carried out at room temperature, and
the chemical shifts are reported as parts per million (ppm) relative to
the resonance of CDCl3 (7.26 ppm in the 1H, 77.0 ppm for the central
line of the triplet in the 13C modes). Low-resolution and high-
resolution mass spectra were obtained on a Micromass Q-TOF-2 by
electron spray ionization (ESI) mode or on a Finnigan MAT95 ST by
electron ionization (EI) mode. Melting points were measured using an
Electrothermal IA9100 digital melting point apparatus and were
uncorrected. All reagents and solvents were reagent grade and were
used without further purification unless otherwise stated. The plates
used for thin-layer chromatography (TLC) were E. Merck silica gel
60F254 (0.25 mm thickness), and they were visualized under short
(254 nm) and long (365 nm) UV light. Chromatographic purifications
were carried out using MN silica gel 60 (230−400 mesh). Substituted
4′-hydroxyflavones 11a−f were prepared as we reported previously.35
Ditosylate 12 was prepared according to the literature report.47,48 The
purity of tested compounds was determined by HPLC, which was
performed by using an Agilent 1100 series installed with an analytic
column of Agilent Prep-Sil Scalar column (4.6 mm × 250 mm, 5 μm)
at UV detection of 320 nm (reference at 450 nm) with isocratic
elution of hexane (50%)/ethyl acetate (25%)/methanol (25%) at a
flow rate of 1 mL/min. All tested compounds were shown to >95%
purity according to HPLC.
General Procedure I for the Preparation of Flavonoid
Dimers 13a−f. A round-bottom flask was charged with correspond-
ing 4′-hydroxyflavones 11a−f (2 equiv), ditosylate 12 (1 equiv),
K2CO3 (2.5 equiv), and DMF. The reaction mixture was stirred at
refluxing temperature for 2 h. When TLC indicated complete
consumption of starting material, the reaction mixture was poured
into a separating funnel containing water. The mixture was
continuously extracted with CH2Cl2. If the mixture could not be
separated into two layers, small amount of 1 M HCl was added. The
combined organic layers were dried over MgSO4, filtered, and
evaporated to give a brown crude reaction mixture. Purification of
the flavonoid dimers was performed by flash column chromatography
on silica gel with 10−20% acetone in CH2Cl2 as eluent to furnish the
desired product.
Figure 5. Intracellular accumulation of 18 in LCC6 and LCC6MDR
cells. LCC6 and LCC6MDR cells were incubated with 10 or 100 μM
18 for 120 min at 37 °C. After the incubation period, cells were lysed
and the supernatant was measured for the concentration of 18 by
HPLC. N = 1−2 independent experiments. The results are presented
as the mean standard error of mean.
The above results suggest that 18 directly interacts with the
substrate recognition site of P-gp and competitively interferes
with substrate binding. This inhibition restores intracellular
accumulation of the substrate and effectively reverses P-gp-
mediated MDR.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(tert-butyloxy-
carbonyl)-1,4,10,13-tetraoxa-7-azatridecane (13a). The titled
compound 13a was obtained from flavone 11a (4.8 g, 20 mmol),
ditosylate 12 (6.1 g, 10 mmol), K2CO3 (3.5 g), and DMF (30 mL) as a
white foam (5.2 g, 70%) according to the general procedure described
CONCLUSIONS
■
1
above: H NMR (CDCl3) δ 1.42 (s, 9H), 3.45 (s, 4H), 3.62 (s, 4H),
In summary, we observed a great improvement of the reversal
potency of P-gp-mediated MDR when flavonoid dimers possess
a substituted amine linker. The most potent amine linked
3.69 (s, 4H), 4.08 (s, 4H), 6.60 (s, 2H), 6.94 (d, J = 8.4 Hz, 4H), 7.30
(d, J = 6.4 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 6.4 Hz, 2H),
7.75 (d, J = 8.4 Hz, 4H), 8.12 (d, J = 7.2 Hz, 2H); 13C NMR (CDCl3)
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δ 28.4, 47.6, 47.9, 67.5, 69.1, 69.2, 69.9, 70.1, 79.6, 106.0, 114.9, 117.9,
123.8, 123.9, 125.0, 125.4, 127.8, 133.5, 155.4, 156.0, 161.5, 163.1,
178.1; LRMS (ESI) m/z 734 (M+ + H, 77), 756 (M+ + Na, 100);
HRMS (ESI) calcd for C43H44NO10 (M+ + H) 734.2965, found
734.2979.
8.8 Hz, 2H); 13C NMR (CDCl3) δ 28.4, 47.7, 47.9, 67.5, 69.1, 69.3,
69.9, 70.1, 73.8, 79.6, 105.4, 110.6 (d, J = 23.6 Hz, C5), 115.1, 119.9
(d, J = 7.8 Hz, C10), 121.7 (d, J = 25.2 Hz, C7), 123.8, 125.0 (d, J =
7.8 Hz, C8), 127.9, 152.2 (d, J = 1.5 Hz, C9), 159.5 (d, J = 245.3 Hz,
C6), 161.7, 163.5, 177.4; LRMS (ESI) m/z 770 (M+ + H, 100), 792
(M+ + Na, 68); HRMS (ESI) calcd for C43H42NO10F2 (M+ + H)
770.2777, found 770.2807.
General Procedure II for the Preparation of Flavonoid
Dimers 14a−f. A round-bottom flask was charged with N-Boc
protected flavonoid dimers 13 and CH2Cl2. The solution was cooled
to 0 °C using an ice bath. An equal volume of TFA was then added
dropwise, and the reaction mixture was stirred vigorously at 0 °C for
1 h and at room temperature for another 1 h. After the mixture was
stirred, the reaction was quenched by pouring the mixture into a
conical flask containing water. The resultant mixture was basified
to pH 10 by using potassium hydroxide solution. The mixture was
continuously extracted with CH2Cl2. The combined organic layers
were dried over MgSO4, filtered, and evaporated to give the desired
product.
1,13-Bis[4′-((3-methoxy)-4H-chromen-4-on-2-yl)phenyl]-7-
(tert-butyloxycarbonyl)-1,4,10,13-tetraoxa-7-azatridecane
(13b). The titled compound 13b was obtained from 4′-hydroxyflavone
11b (0.18 g, 0.67 mmol), ditosylate 12 (0.20 g, 0.33 mmol), K2CO3
(0.12 g), and DMF (6 mL) as a white foam (0.14 g, 53%) according to
the general procedure I described above: 1H NMR (CDCl3) δ 1.40 (s,
9H), 3.45 (s, 4H), 3.61 (s, 4H), 3.79 (s, 4H), 3.87 (s, 6H), 4.13 (s,
4H), 6.63 (s, 2H), 6.89 (d, J = 6.4 Hz, 2H), 7.30 (d, J = 6.4 Hz, 2H),
7.43 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 6.4 Hz, 2H), 7.75 (d, J = 8.4 Hz,
4H), 8.12 (d, J = 7.2 Hz, 2H); 13C NMR (CDCl3) δ 28.4, 47.6, 47.9,
56.0, 68.4, 69.0, 69.2, 69.9, 70.2, 79.5, 106.3, 109.1, 112.8, 117.9, 119.7,
123.7, 124.3, 125.0, 125.4, 133.5, 149.5, 151.4, 155.4, 156.0, 163.1,
178.1; LRMS (ESI) m/z 794 (M+ + H, 27), 816 (M+ + Na, 100);
HRMS (ESI) calcd for C45H48NO12 (M+ + H) 794.3177, found
794.3170.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-1,4,10,13-tetraoxa-
7-azatridecane (14a). The titled compound 14a was obtained from
13a (5.10 g, 6.95 mmol), TFA (20 mL), and CH2Cl2 (20 mL) as a
pale brown oil (4.30 g, 98%) according to the general procedure II
described above: 1H NMR (CDCl3) δ 1.95 (br, 1H), 2.83−2.87
(m, 4H), 3.65−3.69 (m, 4H), 3.82 (t, J = 4.8 Hz, 4H), 4.14 (t, J = 4.8
Hz, 4H), 6.66 (s, 2H), 6.97 (d, J = 8.6 Hz, 4H), 7.34 (dd, J = 7.2, 8.0
Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.61 (dd, J = 7.2, 8.0 Hz, 2H), 7.80
(d, J = 8.6 Hz, 4H), 8.15 (dd, J = 1.2, 8.0 Hz, 2H); 13C NMR (CDCl3)
δ 49.2, 67.5, 69.3, 70.9, 106.1, 114.9, 117.9, 123.8, 124.0, 125.0, 125.5,
127.8, 133.5, 156.0, 161.5, 163.1, 178.2; LRMS (ESI) m/z 634 (M+ +
H, 100); HRMS (ESI) calcd for C38H36NO8 (M+ + H) 634.2441,
found 634.2418.
1,13-Bis[4′-((6-methyl)-4H-chromen-4-on-2-yl)phenyl]-N-
(tert-butyloxycarbonyl)-1,4,10,13-tetraoxa-7-azatridecane
(13c). The titled compound 13c was obtained from flavone 11c
(0.12 g, 0.48 mmol), ditosylate 12 (0.15 g, 0.25 mmol), K2CO3
(0.09 g), and DMF (6 mL) as a white foam (90 mg, 47%) according to
1
the general procedure described above: H NMR (CDCl3) δ 1.42 (s,
9H), 2.36 (s, 6H), 3.46 (t, J = 6.8 Hz, 4H), 3.64 (t, J = 3.6 Hz, 4H), 3.78
(s, 4H), 4.09 (s, 4H), 6.62 (s, 2H), 6.93 (d, J = 8.4 Hz, 4H), 7.31−7.39
(m, 4H), 7.75 (d, J = 8.4 Hz, 4H), 7.89 (s, 2H); 13C NMR (CDCl3) δ
20.9, 28.5, 31.4, 47.8, 48.0, 67.6, 69.1, 70.0, 70.2, 79.7, 105.8, 115.0,
117.7, 123.3, 124.1, 124.9, 128.0, 134.9, 135.1, 154.4, 155.5, 161.7, 162.5,
163.3, 178.3; LRMS (ESI) m/z 762 (M+ + H, 95), 784 (M+ + Na, 100);
HRMS (ESI) calcd for C45H48NO10 (M+ + H) 762.3278, found
762.3289.
1,13-Bis[4′-((5-methoxy)-4H-chromen-4-on-2-yl)phenyl]-7-
(tert-butyloxycarbonyl)-1,4,10,13-tetraoxa-7-azatridecane
(13d). The titled compound 13d was obtained from 4′-hydroxyflavone
11d (80 mg, 0.30 mmol), ditosylate 12 (90 mg, 0.15 mmol), K2CO3
(60 mg), and DMF (5 mL) as a white foam (69 mg, 58%) according
to the general procedure I described above: 1H NMR (CDCl3) δ 1.41
(s, 9H), 3.45 (t, J = 6.8 Hz, 4H), 3.63 (s, 4H), 3.71 (s, 4H), 3.92 (s,
6H), 4.09 (s, 4H), 6.55 (s, 2H), 7.73 (d, J = 7.6 Hz, 2H), 6.93 (d, J =
8.0 Hz, 4H), 7.02 (d, J = 8.0 Hz, 2H), 7.47 (dd, J = 7.6, 8.0 Hz, 2H),
7.74 (d, J = 8.0 Hz, 4H); 13C NMR (CDCl3) δ 28.4, 47.6, 47.9, 56.3,
68.4, 69.0, 69.2, 69.9, 70.1, 79.6, 106.3, 107.5, 110.0, 114.9, 123.7,
127.7, 133.6, 158.1, 159.7, 160.9, 161.3, 178.3; LRMS (ESI) m/z 794
(M+ + H, 35), 816 (M+ + Na, 100); HRMS (ESI) calcd for C45H48
NO12 (M+ + H) 794.3177, found 794.3175.
1,13-Bis[4′-((3-benzyloxy)-4H-chromen-4-on-2-yl)phenyl]-7-
(tert-butyloxycarbonyl)-1,4,10,13-tetraoxa-7-azatridecane
(13e). The titled compound 13e was obtained from 4′-hydroxyflavone
11e (0.19 g, 0.55 mmol), ditosylate 12 (0.17 g, 0.28 mmol), K2CO3
(0.10 g), and DMF (6 mL) as a white foam (0.13 g, 49%) according to
the general procedure I described above: 1H NMR (CDCl3) δ 1.45 (s,
9H), 3.50 (t, J = 6.8 Hz, 4H), 3.60 (s, 4H), 3.79 (s, 4H), 4.10 (s, 4H),
5.07 (s, 4H), 6.91 (d, J = 8.8 Hz, 4H), 7.22−7.41 (m, 14H), 7.55 (dd,
J = 8.0, 8.4 Hz, 2H), 7.98 (d, J = 8.8 Hz, 4H), 8.20 (d, J = 8.0 Hz, 2H);
13C NMR (CDCl3) δ 28.4, 47.7, 47.9, 67.5, 69.1, 69.3, 69.9, 70.1, 73.8,
79.6, 114.3, 117.9, 123.3, 124.0, 124.5, 125.5, 128.0, 128.2, 128.7,
129.0, 130.4, 133.2, 136.7, 139.2, 155.0, 155.4, 156.0, 160.6, 174.8;
LRMS (ESI) m/z 946 (M+ + H, 25), 968 (M+ + Na, 100); HRMS
(ESI) calcd for C57H56NO12 (M+ + H) 946.3803, found 946.3838.
1,13-Bis[4′-((6-fluoro)-4H-chromen-4-on-2-yl)phenyl]-7-
(tert-butyloxycarbonyl)-1,4,10,13-tetraoxa-7-azatridecane
(13f). The titled compound 13f was obtained from 4′-hydroxyflavone
11f (3.09 g, 12.1 mmol), ditosylate 12 (3.60 g, 5.98 mmol), K2CO3
(1.74 g), and DMF (20 mL) as a white foam (2.41 g, 52%) according
to the general procedure I described above: 1H NMR (CDCl3) δ 1.47
(s, 9H), 3.51 (s, 4H), 3.69 (s, 4H), 3.84 (s, 4H), 4.17 (s, 4H), 6.68 9s,
2H), 7.00 (d, J = 8.8 Hz, 4H), 7.39 (ddd, J = 2.0, 8.4, 8.4 Hz, 2H), 7.50
(dd, J = 4.4, 9.2 Hz, 2 H), 7.79 (d, J = 8.8 Hz, 4H), 7.81 (dd, J = 4.4,
1,13-Bis[4′-((3-methoxy)-4H-chromen-4-on-2-yl)phenyl]-
1,4,10,13-tetraoxa-7-azatridecane (14b). The titled compound
14b was obtained from 13b (0.12 g, 0.15 mmol), TFA (4 mL), and
CH2Cl2 (4 mL) as a pale brown oil (95 mg, 91%) according to the
1
general procedure II described above: H NMR (CDCl3) δ 2.13 (br,
1H), 2.79−2.82 (m, 4H), 3.63−3.65 (m, 4H), 3.83−3.84 (m, 4H),
3.89 (s, 6H), 4.16−4.19 (m, 4H), 6.65 (s, 1H), 6.66 (s, 1H), 6.91−
6.94 (m, 2H), 7.29−7.33 (m, 4H), 7.42−7.48 (m, 4H), 7.52−7.58 (m,
2H), 8.11−8.13 (m, 2H); 13C NMR (CDCl3) δ 49.1, 56.0, 68.3, 69.2,
70.9, 79.6, 106.3, 109.2, 112.8, 117.9, 119.7, 123.8, 124.3, 125.0, 125.5,
133.5, 149.5, 151.3, 156.0, 163.1, 178.2; LRMS (ESI) m/z 694 (M+ +
H, 100), 716 (M+ + Na, 19); HRMS (ESI) calcd for C40H40NO10
(M+ + H) 694.2652, found 694.2640.
1,13-Bis[4′-((6-methyl)-4H-chromen-4-on-2-yl)phenyl]-
1,4,10,13-tetraoxa-7-azatridecane (14c). The titled compound
14c was obtained from 13c (72 mg, 0.09 mmol), TFA (4 mL), and
CH2Cl2 (4 mL) as a pale brown oil (56 mg, 90%) according to the
1
general procedure II described above: H NMR (CDCl3) δ 2.03 (br,
1H), 2.41 (s, 6 h), 2.85 (t, J = 4.6 Hz, 4H), 3.68 (t, J = 4.2 Hz, 4H),
3.83 (t, J = 4.6 Hz, 4H), 4.14 (t, J = 4.6 Hz, 4H), 6.66 (s, 2H), 6.97 (d,
J = 8.8 Hz, 4H), 7.36 (d, J = 8.4 Hz, 2H), 7.43 (dd, J = 2.0, 8.0 Hz,
2H), 7.81 (d, J = 8.4 Hz, 4H), 7.93 (s, 2H); 13C NMR (CDCl3) δ 20.9,
48.9, 67.5, 69.4, 70.2, 105.9, 114.9, 117.6, 123.4, 124.1, 124.9, 127.8,
134.8, 134.9, 154.3, 161.5, 163.1, 178.4; LRMS (ESI) m/z 662 (M+ + H,
100), 684 (M+ + Na, 5); HRMS (ESI) calcd for C40H40NO8 (M+ + H)
662.2754, found 662.2758.
1,13-Bis[4′-((5-methoxy)-4H-chromen-4-on-2-yl)phenyl]-
1,4,10,13-tetraoxa-7-azatridecane (14d). The titled compound
14d was obtained from 13d (62 mg, 0.08 mmol), TFA (4 mL), and
CH2Cl2 (4 mL) as a pale brown oil (47 mg, 87%) according to the
1
general procedure II described above: H NMR (CDCl3) δ 2.19 (br,
1H), 2.80 (t, J = 4.6 Hz, 4H), 3.62 (t, J = 4.6 Hz, 4H), 3.77 (t, J = 4.2
Hz, 4H), 3.91 (s, 6H), 4.08 (t, J = 4.6 Hz, 4H), 6.53 (s, 2H), 6.71 (d,
J = 8.0 Hz, 2H), 6.91 (d, J = 8.8 Hz, 4H), 6.99 (d, J = 8.4 Hz, 2H),
7.45 (dd, J = 8.0, 8.4 Hz, 2H), 7.70 (d, J = 8.8 Hz, 4H); 13C NMR
(CDCl3) δ 48.9, 56.5, 67.5, 69.4, 70.2, 106.4, 107.7, 110.1, 114.5,
115.0, 123.9, 127.7, 133.6, 158.2, 159.7, 161.0, 161.3, 178.3; LRMS
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(ESI) m/z 694 (M+ + H, 100), 716 (M+ + Na, 20); HRMS (ESI) calcd
for C40H40NO10 (M+ + H) 694.2652, found 694.2653.
117.9, 123.7, 123.9, 125.0, 125.4, 127.8, 133.5, 156.0, 161.5, 163.1, 178.1;
LRMS (ESI) m/z 677 (M+ + H, 100); HRMS (ESI) calcd for
C40H40NO9(M+ + H) 677.7390, found 677.7382.
1,13-Bis[4′-((3-benzyloxy)-4H-chromen-4-on-2-yl)phenyl]-
1,4,10,13-tetraoxa-7-azatridecane (14e). The titled compound
14e was obtained from 13e (90 mg, 0.10 mmol), TFA (4 mL), and
CH2Cl2 (4 mL) as a pale brown oil (75 mg, 93%) according to the
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(3-ethoxy-3-
oxopropyl)-1,4,10,13-tetraoxa-7-azatridecane (17). A round-
bottom flask was charged with compound 14a (90 mg, 0.14 mmol),
ethyl 3-bromopropionate (40 mg, 0.22 mmol), K2CO3 (40 mg), and
ACN (15 mL). The reaction mixture was stirred at refluxing tem-
perature for 3 h. When TLC indicated complete consumption of
starting material, the reaction mixture was poured into a separating
funnel containing water. The mixture was continuously extracted with
CH2Cl2. The combined organic layers were dried over MgSO4,
filtered, and evaporated to give a crude reaction mixture, which was
subjected to purification by flash column chromatography on silica gel
with 10−20% acetone in CH2Cl2 as eluent to furnish the titled com-
pound 17 as a pale brown oil (65 mg, 62%): 1H NMR (CDCl3) δ 1.23
(t, J = 6.8 Hz, 3H), 2.48 (t, J = 6.8 Hz, 2H), 2.79 (t, J = 6.0 Hz, 4H),
2.94 (t, J = 7.2 Hz, 2H), 3.64 (t, J = 6.0 Hz, 4H), 3.83 (t, J = 4.8 Hz,
4H), 4.08−4.17 (m, 6H), 6.71 (s, 2H), 7.01 (d, J = 8.8 Hz, 4H), 7.38
(dd, J = 7.6, 7.6 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.65 (dd, J = 8.0,
8.4 Hz, 2H), 7.84 (d, J = 8.8 Hz, 4H), 8.19 (d, J = 8.0 Hz, 2H); 13C
NMR (CDCl3) δ 14.2, 50.7, 54.0, 60.4, 67.6, 69.4, 106.2, 115.0, 117.9,
123.9, 124.2, 125.1, 125.6, 128.0, 133.6, 156.1, 161.6, 163.3, 178.3;
LRMS (ESI) m/z 733 (M+ + H, 100); HRMS (ESI) calcd for C43H44
NO10 (M+ + H) 733.8023, found 733.8016.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(benzyl)-
1,4,10,13-tetraoxa-7-azatridecane (18). A round-bottom flask was
charged with compound 14a (1.30 g, 2.05 mmol), benzyl bromide
(0.45 g, 2.63 mmol), K2CO3 (0.40 g), and ACN (40 mL). The
reaction mixture was stirred at refluxing temperature for 3 h. When
TLC indicated complete consumption of starting material, the hot
reaction mixture was filtered to remove solid K2CO3. The filtrate was
cooled in an ice bath, and a large amount of white precipitate was
formed. The titled compound 18 was obtained after suction filtration
as a white solid (1.05 g, 71%): mp 112−114 °C; 1H NMR (CDCl3) δ
2.80 (t, J = 6.0 Hz, 4H), 3.64 (t, J = 6.0 Hz, 4H), 3.72 (s, 2H), 3.78 (t,
J = 4.8 Hz, 4H), 4.11 (t, J = 4.8 Hz, 4H), 6.67 (s, 2H), 6.97 (d, J = 8.8
Hz, 4H), 7.25−7.36 (m, 7H), 7.48 (d, J = 8.4 Hz, 2H), 7.62 (dd, J =
8.0, 8.4 Hz, 2H), 7.79 (d, J = 8.8 Hz, 4H), 8.17 (d, J = 8.0 Hz, 2H);
13C NMR (CDCl3) δ 53.9, 59.8, 67.6, 69.2, 70.2, 106.1, 115.0, 117.9,
1
general procedure II described above: H NMR (CDCl3) δ 2.19 (br,
1H), 2.86 (t, J = 4.6 Hz, 4H), 3.67 (t, J = 4.2 Hz, 4H), 3.82 (t, J = 4.6
Hz, 4H), 4.13 (t, J = 4.6 Hz, 4H), 5.08 (s, 4H), 6.92 (d, J = 9.2 Hz,
4H), 7.22−7.36 (m, 12H), 7.42 (d, J = 8.4 Hz, 2H), 7.58 (dd, J = 8.0,
8.4 Hz, 2H), 7.98 (d, J = 8.8 Hz, 4H), 8.22 (d, J = 8.0 Hz, 2H); 13C
NMR (CDCl3) δ 49.2, 67.4, 69.3, 70.9, 73.8, 114.3, 117.9, 123.4,
124.1, 124.5, 125.6, 128.0, 128.2, 128.8, 130.4, 133.2, 136.7, 139.2,
155.0, 156.1, 160.6, 174.8; LRMS (ESI) m/z 846 (M+ + H, 100), 868
(M+ + Na, 8); HRMS (ESI) calcd for C52H48NO10 (M+ + H)
846.3278, found 846.3268.
1,13-Bis[4′-((6-fluoro)-4H-chromen-4-on-2-yl)phenyl]-
1,4,10,13-tetraoxa-7-azatridecane (14f). The titled compound 14f
was obtained from 13f (2.30 g, 2.99 mmol), TFA (10 mL), and
CH2Cl2 (10 mL) as a pale brown oil (1.99 g, 99%) according to the
1
general procedure II described above: H NMR (CDCl3) δ 2.48 (br,
1H), 2.91 (t, J = 4.6 Hz, 4H), 3.72 (t, J = 4.6 Hz, 4H), 3.87 (t, J = 4.6
Hz, 4H), 4.18 (t, J = 4.6 Hz, 4H), 6.68 (s, 2H), 7.00 (d, J = 8.8 Hz,
4H), 7.39 (ddd, J = 2.0, 8.4, 8.4 Hz, 2H), 7.50 (dd, J = 4.4, 9.2 Hz, 2
H), 7.79 (d, J = 8.8 Hz, 4H), 7.81 (dd, J = 4.4, 8.8 Hz, 2H); 13C NMR
(CDCl3) δ 49.3, 67.6, 69.3, 70.7, 105.4, 110.6 (d, J = 23.6 Hz, C5),
115.1, 119.9 (d, J = 7.8 Hz, C10), 121.7 (d, J = 25.2 Hz, C7), 123.8,
125.0 (d, J = 7.8 Hz, C8), 127.9, 152.2 (d, J = 1.5 Hz, C9), 159.5 (d,
J = 245.3 Hz, C6), 161.7, 163.5, 177.4; LRMS (ESI) m/z 670 (M+ +
H, 100), 692 (M+ + Na, 13); HRMS (ESI) calcd for C38H34NO8F2
(M+ + H) 670.2252, found 670.2232.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(ethyl)-
1,4,10,13-tetraoxa-7-azatridecane (15). A round-bottom flask
was charged with compound 14a (80 mg, 0.13 mmol), bromoethane
(25 mg, 0.23 mmol), K2CO3 (40 mg), and ACN (10 mL). The
reaction mixture was stirred at refluxing temperature for 3 h. When
TLC indicated complete consumption of starting material, the reaction
mixture was poured into a separating funnel containing water. The
mixture was continuously extracted with CH2Cl2. The combined
organic layers were dried over MgSO4, filtered, and evaporated to give
a crude reaction mixture, which was subjected to purification by flash
column chromatography on silica gel with 10−20% acetone in CH2Cl2
as eluent to furnish the titled compound 15 as a pale brown oil (56
123.8, 124.0, 125.0, 125.5, 126.9, 127.8, 128.1, 128.7, 133.5, 139.6,
156.0, 161.6, 163.2, 178.2; LRMS (ESI) m/z 724 (M+ + H, 100), 746
(M+ + Na, 14); HRMS (ESI) calcd for C45H42NO8 (M+ + H)
724.2910, found 724.2917.
1
mg, 67%): H NMR (CDCl3) δ 1.08 (t, J = 7.2 Hz, 3H), 2.77 (q, J =
5.2 Hz, 2H), 2.84 (t, J = 5.2 Hz, 4H), 3.65 (t, J = 4.8 Hz, 4H), 3.82 (t,
J = 4.8 Hz, 4H), 4.15 (t, J = 4.8 Hz, 4H), 6.68 (s, 2H), 6.99 (d, J = 8.8
Hz, 4H), 7.36 (dd, J = 7.6, 7.6 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.65
(dd, J = 8.0, 8.4 Hz, 2H), 7.82 (d, J = 8.8 Hz, 4H), 8.17 (d, J = 8.0 Hz,
2H); 13C NMR (CDCl3) δ 49.2, 53.3, 67.6, 69.3, 69.5, 106.1, 114.9,
117.9, 123.8, 124.1, 125.0, 125.5, 127.9, 133.5, 156.1, 161.5, 163.2,
178.3; LRMS (ESI) m/z 661 (M+ + H, 100); HRMS (ESI) calcd for
C40H40NO8 (M+ + H) 661.7396, found 661.7386.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(2-hydroxyethyl)-
1,4,10,13-tetraoxa-7-azatridecane (16). A round-bottom flask was
charged with compound 14a (70 mg, 0.11 mmol), bromoethanol
(25 mg, 0.20 mmol), K2CO3 (35 mg), and ACN (15 mL). The
reaction mixture was stirred at refluxing temperature for 3 h. When
TLC indicated complete consumption of starting material, the reaction
mixture was poured into a separating funnel containing water. The
mixture was continuously extracted with CH2Cl2. The combined
organic layers were dried over MgSO4, filtered, and evaporated to give
a crude reaction mixture, which was subjected to purification by flash
column chromatography on silica gel with 10−20% acetone in CH2Cl2 as
eluent to furnish the titled compound 16 as a pale brown oil (45 mg,
56%): 1H NMR (CDCl3) δ 2.17 (br, OH), 2.75 (t, J = 5.2 Hz, 2H), 2.84
(t, J = 5.2 Hz, 4H), 3.58 (t, J = 5.2 Hz, 2H), 3.65 (t, J = 4.8 Hz, 4H), 3.84
(t, J = 4.8 Hz, 4H), 4.15 (t, J = 4.8 Hz, 4H), 6.66 (s, 2H), 7.02 (d, J = 8.8
Hz, 4H), 7.39 (dd, J = 7.6, 7.6 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.65
(dd, J = 8.0, 8.4 Hz, 2H), 7.84 (d, J = 8.8 Hz, 4H), 8.19 (d, J = 8.0 Hz,
2H); 13C NMR (CDCl3) δ 54.3, 56.7, 59.3, 67.5, 69.2, 69.8, 105.9, 114.9,
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(4-nitroben-
zyl)-1,4,10,13-tetraoxa-7-azatridecane (19). A round-bottom
flask was charged with compound 14a (100 mg, 0.16 mmol), 4-
nitrobenzyl bromide (52 mg, 0.24 mmol), K2CO3 (45 mg), and ACN
(15 mL). The reaction mixture was stirred at refluxing temperature for
3 h. When TLC indicated complete consumption of starting material,
the reaction mixture was poured into a separating funnel containing
water. The mixture was continuously extracted with CH2Cl2. The
combined organic layers were dried over MgSO4, filtered, and
evaporated to give a crude reaction mixture, which was subjected to
purification by flash column chromatography on silica gel with 10−
20% acetone in CH2Cl2 as eluent to furnish the titled compound 19 as
1
pale brown solid (85 mg, 70%): mp 95−97 °C; H NMR (CDCl3) δ
2.82 (t, J = 5.6 Hz, 4H), 3.64 (t, J = 6.0 Hz, 4H), 3.78 (t, J = 4.8 Hz,
4H), 3.85 (s, 2H), 4.14 (t, J = 4.8 Hz, 4H), 6.70 (s, 2H), 6.99 (d, J =
8.8 Hz, 4H), 7.35 (d, J = 5.2 Hz, 2H), 7.49 (dd, J = 8.0, 8.4 Hz, 4H),
7.65 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.8 Hz, 4H), 8.11 (d, J = 8.0 Hz,
2H), 8.18 (d, J = 7.6 Hz, 2H); 13C NMR (CDCl3) δ 54.1, 59.1, 67.6,
69.3, 70.1, 106.1, 114.9, 117.9, 123.4, 123.8, 124.2, 125.0, 125.6, 127.9,
129.0, 133.6, 146.9, 148.2, 156.1, 161.5, 163.2, 178.3; LRMS (ESI) m/
z 769 (M+ + H, 100), 791 (M+ + Na, 13); HRMS (ESI) calcd for
C45H41N2O10 (M+ + H) 769.2761, found 769.2762.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(4-methoxy-
carbonylbenzyl)-1,4,10,13-tetraoxa-7-azatridecane (20). A
round-bottom flask was charged with compound 14a (120 mg, 0.19
mmol), methyl 4-(bromomethyl)benzoate (60 mg, 0.26 mmol),
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K2CO3 (50 mg), and ACN (15 mL). The reaction mixture was stirred
at refluxing temperature for 3 h. When TLC indicated complete
consumption of starting material, the reaction mixture was poured into
a separating funnel containing water. The mixture was continuously
extracted with CH2Cl2. The combined organic layers were dried over
MgSO4, filtered, and evaporated to give a crude reaction mixture,
which was subjected to purification by flash column chromatography
on silica gel with 10−20% acetone in CH2Cl2 as eluent to furnish the
mixture was continuously extracted with CH2Cl2. The combined organic
layers were dried over MgSO4, filtered, and evaporated to give a crude
reaction mixture, which was subjected to purification by flash column
chromatography on silica gel with 10−20% acetone in CH2Cl2 as eluent to
furnish the titled compound 23 as a pale brown oil (72 mg, 63%):
1H NMR (CDCl3) δ 2.88 (s, 6H), 3.63 (t, J = 5.2 Hz, 4H), 3.78 (t, J =
4.8 Hz, 6H), 4.11 (t, J = 4.4 Hz, 4H), 6.68 (s, 2H), 6.99 (d, J = 8.8 Hz,
4H), 7.37 (dd, J = 8.0, 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.63−
7.67 (m, 4H), 7.77−7.81 (m, 6H), 8.19 (dd, J = 1.2, 8.4 Hz, 2H); 13C
NMR (CDCl3) δ 52.5, 54.2, 67.6, 69.3, 70.1, 106.1, 115.0, 117.9, 123.1,
123.9, 124.0, 125.0, 125.6, 127.9, 132.1, 133.5, 133.8, 156.1, 161.6,
163.2, 168.3, 178.3; LRMS (ESI) m/z 807 (M+ + H, 20), 829 (M+ +
Na, 4); HRMS (ESI) calcd for C48H43N2O10 (M+ + H) 807.2918, found
807.2920.
1
titled compound 20 as a pale brown oil (98 mg, 66%): H NMR
(CDCl3) δ 2.90 (t, J = 5.6 Hz, 4H), 3.67 (t, J = 6.0 Hz, 4H), 3.78 (t, J =
4.8 Hz, 4H), 3.82 (s, 2H), 3.87 (s, 3H), 4.14 (t, J = 4.8 Hz, 4H), 6.70 (s,
2H), 6.99 (d, J = 8.8 Hz, 4H), 7.37 (dd, J = 8.0, 8.4 Hz, 2H), 7.49 (d, J =
8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.65 (dd, J = 8.0, 8.4 Hz, 2H), 7.83
(d, J = 8.8 Hz, 4H), 7.95 (d, J = 8.0 Hz, 2H), 8.18 (d, J = 7.6 Hz, 2H);
13C NMR (CDCl3) δ 54.0, 67.6, 69.2, 106.1, 115.0, 117.9, 123.8, 124.1,
125.0, 125.6, 127.9, 128.6, 129.5, 133.5, 156.1, 161.5, 163.2, 178.3; LRMS
(ESI) m/z 782 (M+ + H, 100), 804 (M+ + Na, 8); HRMS (ESI) calcd for
C47H44NO10 (M+ + H) 782.2965, found 782.2959.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(3-phenyl-
propyl)-1,4,10,13-tetraoxa-7-azatridecane (24). A round-bottom
flask was charged with compound 14a (110 mg, 0.17 mmol), 3-bromo-
1-phenylpropane (60 mg, 0.30 mmol), K2CO3 (50 mg), and ACN
(15 mL). The reaction mixture was stirred at refluxing temperature for
3 h. When TLC indicated complete consumption of starting material,
the reaction mixture was poured into a separating funnel containing
water. The mixture was continuously extracted with CH2Cl2. The
combined organic layers were dried over MgSO4, filtered, and
evaporated to give a crude reaction mixture, which was subjected to
purification by flash column chromatography on silica gel with 10−
20% acetone in CH2Cl2 as eluent to furnish the titled compound 24 as
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(4-pyridyl-
methyl)-1,4,10,13-tetraoxa-7-azatridecane (21). A round-bottom
flask was charged with compound 14a (210 mg, 0.33 mmol), 4-
(bromomethyl)pyridine hydrobromide salt (85 mg, 0.34 mmol),
K2CO3 (110 mg), and ACN (15 mL). The reaction mixture was
stirred at refluxing temperature for 3 h. When TLC indicated complete
consumption of starting material, the reaction mixture was poured into
a separating funnel containing water. The mixture was continuously
extracted with CH2Cl2. The combined organic layers were dried over
MgSO4, filtered, and evaporated to give a crude reaction mixture, which
was subjected to purification by flash column chromatography on silica gel
with 10−20% acetone in CH2Cl2 as eluent to furnish the titled compound
21 as a pale brown oil (130 mg, 54%): 1H NMR (CDCl3) δ 2.81 (t, J =
5.6 Hz, 4H), 3.63 (t, J = 6.0 Hz, 4H), 3.78 (t, J = 4.8 Hz, 4H), 3.84 (s,
2H), 4.14 (t, J = 4.8 Hz, 4H), 6.70 (s, 2H), 6.99 (d, J = 8.8 Hz, 4H), 7.30
(d, J = 5.2 Hz, 2H), 7.37 (dd, J = 8.0, 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz,
2H), 7.62 (dd, J = 8.0, 8.4 Hz, 2H), 7.83 (d, J = 8.8 Hz, 4H), 8.17 (d, J =
8.0 Hz, 2H), 8.49 (d, J = 5.2 Hz, 2H); 13C NMR (CDCl3) δ 54.2, 58.7,
67.6, 69.3, 70.0, 106.2, 115.0, 117.9, 123.7, 123.9, 124.2, 125.1, 125.6,
127.9, 133.6, 149.6, 156.1, 161.5, 163.2, 178.3; LRMS (ESI) m/z 725 (M+
+ H, 25); HRMS (ESI) calcd for C44H41N2O8 (M+ + H) 725.2863, found
725.2859.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(2-pyridyl-
methyl)-1,4,10,13-tetraoxa-7-azatridecane (22). A round-bottom
flask was charged with compound 14a (110 mg, 0.17 mmol), 2-
(bromomethyl)pyridine hydrobromide salt (49 mg, 0.19 mmol),
K2CO3 (60 mg), and ACN (10 mL). The reaction mixture was stirred
at refluxing temperature for 3 h. When TLC indicated complete
consumption of starting material, the reaction mixture was poured into
a separating funnel containing water. The mixture was continuously
extracted with CH2Cl2. The combined organic layers were dried over
MgSO4, filtered, and evaporated to give a crude reaction mixture,
which was subjected to purification by flash column chromatography
on silica gel with 10−20% acetone in CH2Cl2 as eluent to furnish the
titled compound 22 as an off white solid (73 mg, 58%): mp 107−
109 °C; 1H NMR (CDCl3) δ 2.87 (t, J = 5.6 Hz, 4H), 3.66 (t, J = 6.0 Hz,
4H), 3.78 (t, J = 4.8 Hz, 4H), 3.89 (s, 2H), 4.14 (t, J = 4.8 Hz, 4H), 6.68
(s, 2H), 6.98 (d, J = 8.8 Hz, 4H), 7.13 (dd, J = 8.0, 8.4 Hz, 1H), 7.47
(dd, J = 8.0, 8.4 Hz, 2H), 7.48−7.63 (m, 6H), 7.81 (d, J = 8.8 Hz, 4H),
8.17 (d, J = 8.8 Hz, 2H), 8.49 (d, J = 8.4 Hz, 1H); 13C NMR (CDCl3) δ
54.2, 61.3, 67.6, 69.2, 70.0, 106.1, 115.0, 117.9, 121.9, 123.0, 123.8, 124.0,
125.0, 125.5, 127.9, 133.5, 136.3, 148.9, 156.1, 160.0, 161.6, 163.2, 178.3;
LRMS (ESI) m/z 725 (M+ + H, 23), 747 (M+ + Na, 8); HRMS (ESI)
calcd for C44H41N2O8 (M+ + H) 725.2863, found 725.2849.
1
a pale brown oil (86 mg, 66%): H NMR (CDCl3) δ 1.81 (m, 2H),
2.58−2.62 (m, 4H), 2.79 (t, J = 5.2 Hz, 4H), 3.63 (t, J = 4.8 Hz, 4H),
3.81 (t, J = 4.8 Hz, 4H), 4.14 (t, J = 4.8 Hz, 4H), 6.70 (s, 2H), 7.00 (d,
J = 8.8 Hz, 4H), 7.15−7.26 (m, 5H), 7.37 (dd, J = 7.6, 7.6 Hz, 2H),
7.51 (d, J = 8.4 Hz, 2H), 7.65 (dd, J = 8.0, 8.4 Hz, 2H), 7.83 (d, J = 8.8
Hz, 4H), 8.19 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3) δ28.8, 33.4,
33.5, 54.1, 54.9, 67.7, 69.3, 70.1, 106.1, 115.0, 117.9, 123.9, 124.1,
125.1, 125.6, 125.8, 127.9, 128.3, 128.4, 133.6, 142.2, 156.1, 161.6, 163.3,
178.3; LRMS (ESI) m/z 752 (M+ + H, 55); HRMS (ESI) calcd for
C47H46NO8 (M+ + H) 751.8621, found 751.8612.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(4-fluoroben-
zyl)-1,4,10,13-tetraoxa-7-azatridecane (25). A round-bottom
flask was charged with compound 14a (0.31 g, 0.49 mmol), 4-
fluorobenzyl methanesufonate (0.12 g, 0.59 mmol), K2CO3 (90 mg),
and ACN (20 mL). The reaction mixture was stirred at refluxing tem-
perature for 3 h. When TLC indicated complete consumption of
starting material, the hot reaction mixture was filtered to remove solid
K2CO3. The filtrate was cooled in an ice bath, and a large amount of
white precipitate was formed. The titled compound 25 was obtained
after suction filtration as a white solid (0.25 g, 69%): mp 119−122 °C;
1H NMR (CDCl3) δ 2.83 (s, 4H), 3.67 (t, J = 4.6 Hz, 4H), 3.79 (t, J =
4.6 Hz, 4H), 3.81 (s, 2H), 4.14 (t, J = 4.6 Hz, 4H), 6.69 (s, 2H), 6.98
(d, J = 8.6 Hz, 4H), 7.36 (dd, J = 7.2, 8.0 Hz, 2H), 7.47−7.54 (m, 6H),
7.64 (dd, J = 7.2, 8.0 Hz, 2H), 7.82 (d, J = 8.6 Hz, 4H), 8.16 (d, J = 8.0
Hz, 2H); 13C NMR (CDCl3) δ 54.0, 59.3, 67.7, 69.3, 70.2, 106.1,
115.0, 117.9, 123.9, 124.1, 125.0, 125.1, 125.6, 127.9, 128.8, 133.5,
144.3, 156.1, 161.6, 163.2, 178.2; LRMS (ESI) m/z 742 (M+ + H,
100); HRMS (ESI) calcd for C45H41NO8F (M+ + H) 742.2816, found
742.2787.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(4-trifluorome-
thylbenzyl)-1,4,10,13-tetraoxa-7-azatridecane (26). A round-
bottom flask was charged with compound 14a (210 mg, 0.33 mmol),
4-trifluoromethylbenzyl chloride (90 mg, 0.46 mmol), K2CO3
(70 mg), and ACN (15 mL). The reaction mixture was stirred at
refluxing temperature for 3 h. When TLC indicated complete con-
sumption of starting material, the reaction mixture was poured into a
separating funnel containing water. The mixture was continuously
extracted with CH2Cl2. The combined organic layers were dried over
MgSO4, filtered, and evaporated to give a crude reaction mixture,
which was subjected to purification by flash column chromatography
on silica gel with 10−20% acetone in CH2Cl2 as eluent to furnish the
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-[2-(1,3-dioxo-
1,3-dihydro-2H-isoindol-2-yl)ethyl]-1,4,10,13-tetraoxa-7-
azatridecane (23). A round-bottom flask was charged with
compound 14a (90 mg, 0.14 mmol), 1-bromo-2-(1,3-dioxo-1,3-dihydro-
2H-isoindol-2-yl)ethane (65 mg, 0.26 mmol), K2CO3 (50 mg), and ACN
(10 mL). The reaction mixture was stirred at refluxing temperature for 3 h.
When TLC indicated complete consumption of starting material, the
reaction mixture was poured into a separating funnel containing water. The
1
titled compound 26 as a pale brown oil (190 mg, 72%): H NMR
(CDCl3) δ 2.83 (s, 4H), 3.68 (s, 4H), 3.79 (s, 4H), 3.83 (s, 2H), 4.17
(s, 4H), 6.73 (s, 2H), 6.96−7.03 (m, 6H), 7.34−7.42 (m, 4H), 7.55
2009
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Journal of Medicinal Chemistry
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(d, J = 8.0 Hz, 2H), 7.68 (dd, J = 8.0, 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz,
4H), 8.22 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3) δ 67.7, 69.3, 106.2,
115.0, 117.9, 123.9, 124.2, 125.1, 125.7, 128.0, 133.6, 156.2, 161.6,
163.3, 178.3; LRMS (ESI) m/z 792 (M+ + H, 100); HRMS (ESI)
calcd for C46H41NO8F3 (M+ + H) 792.2784, found 792.2764.
K2CO3. The filtrate was cooled in an ice bath, and a large amount of
white precipitate was formed. The titled compound 30 was obtained
after suction filtration as a white solid (145 mg, 56%): mp 78−80 °C;
1H NMR (CDCl3) δ 2.83 (s, 4H), 3.68 (s, 4H), 3.73 (s, 2H), 3.83 (s,
4H), 4.17 (s, 4H), 6.72 (s, 2H), 7.01 (d, J = 8.4 Hz, 4H), 7.04 (s, 2H),
7.39 (dd, J = 8.0, 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.67 (dd, J =
8.0, 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 4H), 8.21 (d, J = 8.4 Hz, 2H);
13C NMR (CDCl3) δ 53.9, 58.6, 67.6, 69.4, 70.2, 106.2, 115.0, 117.9,
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(2-fluoroben-
zyl)-1,4,10,13-tetraoxa-7-azatridecane (27). A round-bottom
flask was charged with compound 14a (190 mg, 0.30 mmol), 2-
fluorobenzyl bromide (90 mg, 0.48 mmol), K2CO3 (70 mg), and ACN
(20 mL). The reaction mixture was stirred at refluxing temperature for
3 h. When TLC indicated complete consumption of starting material,
the hot reaction mixture was filtered to remove solid K2CO3. The
filtrate was cooled in an ice bath, and a large amount of white
precipitate was formed. The titled compound 27 was obtained after
123.9, 124.2, 125.1, 125.6, 127.9, 133.6, 156.1, 161.6, 163.2, 178.3;
LRMS (ESI) m/z 778 (M+ + H, 100); HRMS (ESI) calcd for
C45H39NO8F3(M+ + H) 778.2628, found 778.2657.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(tert-butylace-
tyl)-1,4,10,13-tetraoxa-7-azatridecane (31). A round-bottom flask
was charged with compound 14a (100 mg, 0.16 mmol), tert-
butylacetyl chloride (40 mg, 0.30 mmol), and pyridine (10 mL).
The reaction mixture was stirred at room temperature for 2 h. When
TLC indicated complete consumption of starting material, the reaction
mixture was poured into a separating funnel containing 1 M
hydrochloric acid solution. The mixture was continuously extracted
with CH2Cl2. The combined organic layers were dried over MgSO4,
filtered, and evaporated to give a crude reaction mixture, which was
subjected to purification by flash column chromatography on silica gel
with 10−20% acetone in CH2Cl2 as eluent to furnish the titled
compound 31 as a pale brown oil (81 mg, 70%): 1H NMR (CDCl3) δ
1.01 (s, 9H), 3.59 (s, 2H), 3.59−3.71 (m, 8H), 3.79 (t, J = 4.8 Hz,
4H), 4.14 (t, J = 4.8 Hz, 4H), 6.71 (s, 1H), 6.72 (s, 1H), 6.98 (dd, J =
8.0, 8.4 Hz, 4H), 7.34 (dd, J = 7.2, 8.0 Hz, 2H), 7.51 (dd, J = 8.0, 8.4
Hz, 2H), 7.63 (dd, J = 7.2, 8.0 Hz, 2H), 7.82 (d, J = 8.6 Hz, 4H), 8.17
(dd, J = 8.0, 8.4 Hz, 2H); 13C NMR (CDCl3) δ 29.9, 31.4, 44.6, 46.4,
49.5, 67.6, 69.3, 69.6, 69.8, 69.9, 106.1, 106.1, 114.9, 117.9, 117.9,
123.8, 124.1, 124.1, 125.1, 125.6, 127.9, 133.6, 156.1, 161.5, 161.6,
163.2, 163.3, 172.4, 178.2; LRMS (ESI) m/z 732 (M+ + H, 100), 754
(M+ + Na, 90); HRMS (ESI) calcd for C44H46NO9 (M+ + H)
732.3173, found 732.3147.
1
suction filtration as a white solid (153 mg, 69%): mp 82−84 °C; H
NMR (CDCl3) δ 2.87 (s, 4H), 3.71 (s, 4H), 3.82 (s, 4H), 3.84 (s, 2H),
4.15 (t, J = 4.6 Hz, 4H), 6.72 (s, 2H), 7.01−7.23 (m, 7H), 7.39 (dd,
J = 8.0, 8.4 Hz, 2H), 7.49 (m, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.67 (dd,
J = 8.0, 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 4H), 8.18 (d, J = 8.4 Hz, 2H);
13C NMR (CDCl3) δ 52.1, 53.9, 67.7, 69.3, 70.0, 106.1, 115.0, 115.3,
117.9, 123.9, 124.1, 125.1, 125.6, 127.9, 131.4, 133.6, 156.1, 160.1,
161.6, 162.5, 163.3, 178.3; LRMS (ESI) m/z 742 (M+ + H, 100), 764
(M+ + Na, 4); HRMS (ESI) calcd for C45H41NO8F (M+ + H)
742.2816, found 742.2852.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(3-fluoroben-
zyl)-1,4,10,13-tetraoxa-7-azatridecane (28). A round-bottom
flask was charged with compound 14a (190 mg, 0.30 mmol), 3-
fluorobenzyl bromide (90 mg, 0.48 mmol), K2CO3 (70 mg), and ACN
(20 mL). The reaction mixture was stirred at refluxing temperature for
3 h. When TLC indicated complete consumption of starting material,
the hot reaction mixture was filtered to remove solid K2CO3. The
filtrate was cooled in an ice bath, and a large amount of white
precipitate was formed. The titled compound 28 was obtained after
1
suction filtration as a white solid (160 mg, 72%): mp 91−93 °C; H
NMR (CDCl3) δ 2.85 (s, 4H), 3.69 (s, 4H), 3.78 (s, 2H), 3.83 (t, J =
4.6 Hz, 4H), 4.17 (t, J = 4.6 Hz, 4H), 6.72 (s, 2H), 6.94 (dd, J = 8.0,
8.4 Hz, 1H), 7.02 (J = 8.8 Hz, 4H), 7.13−7.24 (m, 3H), 7.39 (dd, J =
8.0, 8.4 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.67 (dd, J = 8.0, 8.4 Hz,
2H), 7.85 (d, J = 8.4 Hz, 4H), 8.21 (d, J = 8.4 Hz, 2H); 13C NMR
(CDCl3) δ 54.0, 59.3, 67.7, 69.3, 70.2, 106.2, 115.0, 117.9, 123.9,
124.2, 125.1, 125.6, 127.9, 129.6, 133.6, 156.1, 161.6, 161.8, 163.3,
164.2, 178.3; LRMS (ESI) m/z 742 (M+ + H, 100), 764 (M+ + Na, 5);
HRMS (ESI) calcd for C45H41NO8F (M+ + H) 742.2816, found
742.2828.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(3,4-difluoro-
benzyl)-1,4,10,13-tetraoxa-7-azatridecane (29). A round-bottom
flask was charged with compound 14a (210 mg, 0.33 mmol), 3,4-
difluorobenzyl bromide (90 mg, 0.43 mmol), K2CO3 (70 mg), and
ACN (20 mL). The reaction mixture was stirred at refluxing
temperature for 3 h. When TLC indicated complete consumption of
starting material, the hot reaction mixture was filtered to remove solid
K2CO3. The filtrate was cooled in an ice bath, and a large amount of
white precipitate was formed. The titled compound 29 was obtained
after suction filtration as a white solid (160 mg, 64%): mp 96−98 °C;
1H NMR (CDCl3) δ 2.83 (s, 4H), 3.67 (s, 4H), 3.72 (s, 2H), 3.82 (t,
J = 4.6 Hz, 4H), 4.16 (t, J = 4.6 Hz, 4H), 6.72 (s, 2H), 6.98−7.06 (m,
6H), 7.28 (m, 1H), 7.39 (dd, J = 8.0, 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz,
2H), 7.67 (dd, J = 8.0, 8.4 Hz, 2H), 7.85 (d, J = 8.8 Hz, 4H), 8.21 (d,
J = 8.4 Hz, 2H); 13C NMR (CDCl3) δ 53.8, 58.7, 67.7, 69.3, 70.2,
106.2, 115.0, 116.6, 116.8, 117.9, 123.9, 124.2, 125.1, 125.6, 127.9,
133.6, 156.1, 161.6, 163.2, 178.3; LRMS (ESI) m/z 760 (M+ + H,
100); HRMS (ESI) calcd for C45H40NO8F2 (M+ + H) 760.2722, found
760.2757.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(3,4,5-trifluoro-
benzyl)-1,4,10,13-tetraoxa-7-azatridecane (30). A round-bottom
flask was charged with compound 14a (210 mg, 0.33 mmol), 3,4,5-
trifluorobenzyl bromide (100 mg, 0.42 mmol), K2CO3 (75 mg), and
ACN (20 mL). The reaction mixture was stirred at refluxing
temperature for 3 h. When TLC indicated complete consumption of
starting material, the hot reaction mixture was filtered to remove solid
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(benzoyl)-
1,4,10,13-tetraoxa-7-azatridecane (32). A round-bottom flask was
charged with compound 14a (90 mg, 0.14 mmol), benzoyl chloride
(42 mg, 0.30 mmol), and pyridine (10 mL). The reaction mixture was
stirred at room temperature for 2 h. When TLC indicated complete
consumption of starting material, the reaction mixture was poured into
a separating funnel containing 1 M hydrochloric acid solution. The
mixture was continuously extracted with CH2Cl2. The combined
organic layers were dried over MgSO4, filtered, and evaporated to give
a crude reaction mixture, which was subjected to purification by flash
column chromatography on silica gel with 10−20% acetone in CH2Cl2
as eluent to furnish the titled compound 32 as a pale brown oil (76
1
mg, 73%): H NMR (CDCl3) δ 3.61 (s, 4H), 3.73 (s, 4H), 3.88 (s,
4H), 4.18 (s, 4H), 6.74 (s, 2H), 7.01 (d, J = 8.8 Hz, 4H), 7.31−7.41
(m, 7H), 7.52 (d, J = 8.4 Hz, 2H), 7.66 (dd, J = 8.0, 8.4 Hz, 2H), 7.84
(d, J = 8.8 Hz, 4H), 8.19 (d, J = 8.4 Hz, 2H); 13C NMR (CDCl3) δ
67.6, 69.4, 69.5, 106.1, 115.0, 118.0, 123.8, 124.1, 125.1, 125.6, 126.8,
128.0, 128.3, 129.3, 133.6, 136.7, 156.1, 161.6, 163.3, 172.4, 178.2;
LRMS (ESI) m/z 738 (M+ + H, 100), 760 (M+ + Na, 91); HRMS
(ESI) calcd for C45H40NO9 (M+ + H) 738.2703, found 738.2681.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(4-hydroxy-
1,4-dioxobutyl)-1,4,10,13-tetraoxa-7-azatridecane (33). A
round-bottom flask was charged with compound 14a (70 mg,
0.11 mmol), succinic anhydride (22 mg, 0.22 mmol), and pyridine
(10 mL). The reaction mixture was stirred at room temperature for 14 h.
When TLC indicated complete consumption of starting material, the
reaction mixture was poured into a separating funnel containing 1 M
hydrochloric acid solution. The mixture was continuously extracted with
CH2Cl2. The combined organic layers were dried over MgSO4, filtered,
and evaporated to give a crude reaction mixture, which was subjected to
purification by flash column chromatography on silica gel with 20−40%
acetone in CH2Cl2 as eluent to furnish the titled compound 33 as a pale
brown oil (48 mg, 59%): 1H NMR (CDCl3) δ 2.61−2.77 (m, 4H), 3.62
(t, J = 5.2 Hz, 4H), 3.69 (t, J = 4.8 Hz, 4H), 3.78 (s, 4H), 4.13 (t, J = 4.8
Hz, 4H), 6.70 (s, 1H), 6.72 (s,1H), 6.99 (d, J = 8.8 Hz, 4H), 7.36 (dd, J =
7.6, 7.6 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.65 (dd, J = 8.0, 8.4 Hz, 2H),
2010
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Journal of Medicinal Chemistry
Article
7.81 (d, J = 8.8 Hz, 4H), 8.16 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3) δ
28.1, 29.7, 46.7, 49.0, 67.5, 69.2, 69.3, 69.6, 105.9, 114.9, 117.9, 123.7,
124.0, 124.1, 125.1, 125.5, 127.9, 133.6, 156.1, 161.4, 161.6, 163.3, 172.8,
175.9, 178.5; LRMS (ESI) m/z 734 (M+ + H, 100), 756 (M+ + Na, 65);
HRMS (ESI) calcd for C42H40NO11(M+ + H) 734.2601, found 734.2602.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-[(S)-2-amino-
1-oxopropyl]-1,4,10,13-tetraoxa-7-azatridecane (34). A stirred
solution of the compound 14a (190 mg, 0.30 mmol) in CH2Cl2
(15 mL) at 0 °C was treated successively with 1-hydroxybenzotriazole
(60 mg, 0.44 mmol), Boc-Ala-OH (68 mg, 0.36 mmol), and dicyclo-
hexylcarbodiimide (82 mg, 0.40 mmol). The mixture was allowed to
warm to room temperature, and stirring was continued for 14 h. The
reaction mixture was then evaporated to give a crude reaction mixture,
which was subjected to purification by flash column chromatography on
silica gel with 10−20% acetone in CH2Cl2 as eluent to furnish the
compound 1,13-bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-[(S)-2-(tert-
butyloxycarbonylamino)-1-oxopropyl]-1,4,10,13-tetraoxa-7-azatridecane
as a white foam (170 mg, 70%): 1H NMR (CDCl3) δ 1.23 (d, J = 6.8 Hz,
3H), 1.35 (s, 9H), 3.45−3.75 (m, 12H), 4.06 (s, 4H), 4.67 (m, 1H), 5.39
(d, J = 8.0 Hz, 1H), 6.62 (s, 1H), 6.63 (s, 1H), 6.92 (dd, J = 8.0, 8.4 Hz,
4H), 7.30 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0
Hz, 2H), 7.75 (d, J = 8.0, 8.4 Hz, 4H), 8.09 (d, J = 8.0 Hz, 2H); 13C
NMR (CDCl3) δ 19.3, 28.3, 46.1, 46.7, 48.6, 67.4, 67.5, 69.3, 69.4, 69.5,
79.4, 105.8, 110.4, 114.9, 117.9, 118.2, 123.6, 123.8, 125.0, 125.2, 125.4,
126.7, 127.9, 128.5, 133.6, 155.0, 155.9, 161.5, 161.5, 163.3, 163.4, 173.7,
178.4; LRMS (ESI) m/z 805 (M+ + H, 100), 827 (M+ + Na, 46); HRMS
(ESI) calcd for C46H49N2O11 (M+ + H) 805.3336, found 805.3340. The
titled compound 34 was then obtained from the compound 1,13-bis[4′-
(4H-chromen-4-on-2-yl)phenyl]-7-[(S)-2-(tert-butyloxycarbobylamino)-
1-oxopropyl]-1,4,10,13-tetraoxa-7-azatridecane (170 mg, 0.21 mmol),
TFA (5 mL), and CH2Cl2 (5 mL) as a pale brown oil (130 mg, 87%)
according to the general procedure II described above: 1H NMR
(CDCl3) δ 1.22 (d, J = 6.8 Hz, 3H), 2.01 (s, NH2), 3.44−3.81 (m, 12H),
3.93 (d, J = 6.8 Hz, 1H), 4.14 (t, J = 4.8 Hz, 4H), 6.67 (s, 1H), 6.68 (s,
1H), 6.98 (dd, J = 8.0, 8.4 Hz, 4H), 7.34 (m, 2H), 7.49 (d, J = 8.0 Hz,
2H), 7.63 (m, 2H), 7.79−7.82 (m, 4H), 8.15 (d, J = 8.0 Hz, 2H); 13C
NMR (CDCl3) δ 21.3, 46.5, 48.4, 67.5, 69.3, 69.4, 69.6, 69.7, 106.1, 114.9,
117.9, 117.9, 123.8, 124.1, 124.2, 125.0, 125.5, 127.9, 133.5, 156.0, 161.3,
161.5, 163.1, 177.1, 178.2; LRMS (ESI) m/z 705 (M+ + H, 100), 727
(M+ + Na, 3); HRMS (ESI) calcd for C41H41N2O9 (M+ + H) 705.2812,
found 705.2815.
CH2Cl2. The combined organic layers were dried over MgSO4,
filtered, and evaporated to give a crude reaction mixture, which was
subjected to purification by flash column chromatography on silica gel
with 10−20% acetone in CH2Cl2 as eluent to furnish the titled com-
pound 36 as a pale brown oil (61 mg, 66%): 1H NMR (CDCl3) δ 2.38
(s, 3H), 3.42 (t, J = 5.2 Hz, 4H), 3.72 (t, J = 4.8 Hz, 4H), 3.77 (t, J =
4.8 Hz, 4H), 4.08 (t, J = 4.8 Hz, 4H), 6.68 (s, 2H), 6.98 (d, J = 8.8 Hz,
4H), 7.25 (d, J = 7.6 Hz, 2H), 7.35 (dd, J = 7.6, 7.6 Hz, 2H), 7.49 (d,
J = 8.4 Hz, 2H), 7.63 (dd, J = 8.0, 8.4 Hz, 2H), 7.71 (d, J = 7.6 Hz,
2H), 7.82 (d, J = 8.8 Hz, 4H), 8.17 (d, J = 8.0 Hz, 2H); 13C NMR
(CDCl3) δ 21.5, 49.0, 67.5, 69.3, 70.5, 106.1, 114.9, 117.9, 123.8,
124.1, 125.0, 125.5, 127.1, 129.6, 133.6, 136.6, 143.3, 156.0, 161.5,
163.1, 178.3; LRMS (ESI) m/z 788 (M+ + H, 55), 810 (M+ + Na, 67);
HRMS (ESI) calcd for C45H42NO10S (M+ + H) 788.2529, found
788.2540.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-7-(dansyl)-
1,4,10,13-tetraoxa-7-azatridecane (37). A round-bottom flask was
charged with compound 14a (120 mg, 0.19 mmol), dansyl chloride
(80 mg, 0.30 mmol), and pyridine (10 mL). The reaction mixture was
stirred at room temperature for 14 h. When TLC indicated complete
consumption of starting material, the reaction mixture was poured into
a separating funnel containing 1 M hydrochloric acid solution. The
mixture was continuously extracted with CH2Cl2. The combined
organic layers were dried over MgSO4, filtered, and evaporated to give
a crude reaction mixture, which was subjected to purification by flash
column chromatography on silica gel with 10−20% acetone in CH2Cl2
as eluent to furnish the titled compound 37 as a pale yellow solid (120
1
mg, 73%): mp 72−74 °C; H NMR (CDCl3) δ 2.81 (s, 6H), 3.64−
3.67 (m, 12H), 3.98 (t, J = 4.8 Hz, 4H), 6.63 (s, 2H), 6.90 (d, J = 8.8
Hz, 4H), 7.12 (d, J = 7.6 Hz, 1H), 7.32 (dd, J = 7.6, 8.0 Hz, 2H),
7.41−7.50 (m, 4H), 7.56 (dd, J = 7.6, 8.0 Hz, 2H), 7.74 (d, J = 8.8 Hz,
4H), 8.10 (d, J = 8.0 Hz, 2H), 8.12 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0
Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H); 13C NMR (CDCl3) δ 45.3, 48.1,
67.4, 69.2, 70.2, 106.0, 114.9, 115.1, 117.9, 119.5, 123.1, 123.7, 123.9,
125.0, 125.4, 127.8, 128.0, 128.6, 130.0, 130.0, 130.2, 133.5, 135.6,
151.7, 156.0, 161.4, 163.1, 178.2; LRMS (ESI) m/z 867 (M+ + H, 85),
889 (M+ + Na, 18); HRMS (ESI) calcd for C50H47N2O10S (M+ + H)
867.2951, found 867.2927.
Materials for Biological Studies. Dimethyl sulfoxide (DMSO),
paclitaxel, DOX, vinblastine, vincristine, verapamil, cyclosporine A,
mitoxantrone, daunorubicin, topotecan, Ko143, and phenazine
methosulfate (PMS) were purchased from Sigma-Aldrich. Dulbecco’s
modified Eagle’s medium (DMEM), Roswell Park Memorial Institute
(RPMI) 1640 medium, trypsin/ethylenediaminetetraacetic acid
(EDTA), and penicillin/streptomycin were purchased from Gibco
BRL. The fetal bovine serum (FBS) was purchased from HyClone
Laboratories. 3-(4,5-Dimethylthiazol-2-yl)-5-[3-(carboxymethoxy)-
phenyl]-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased
from Promega. The human breast cancer cell lines MDA435/LCC6
and MDA435/LCC6MDR were kindly provided by Dr. Robert Clarke
(Georgetown University, U.S.).The human ovarian carcinoma cell lines
2008/P and 2008/MRP1 were generous gifts from Prof. P. Borst (The
Netherlands Cancer Institute, Amsterdam, The Netherlands). The human
embryonic kidney (HEK) 293 cell lines, HEK293/pcDNA3.1 (empty
vector-transfected), and HEK293/R2 (BCRP-transfected) were kindly
provided by Dr. Kenneth To (The Chinese University of Hong Kong,
China). Agilent Prep-SIL Scalar column (440905-901, 4.6 mm × 250
mm, 5 μm) was purchased from Tegent Technology. All other reagents
or solvents used were either analytical or high-performance liquid
chromatography (HPLC) grade and were purchased from Tedia.
Cell Culture. MDA435/LCC6 and MDA435/LCC6MDR cell lines
were cultured in supplemented DMEM with 10% heat inactivated FBS
and 100 U/mL penicillin and 100 μg/mL streptomycin. 2008/P and
2008/MRP1 cells or HEK293/pcDNA3.1 and HEK293/R2 were
cultured in RPMI 1640 medium containing heat inactivated 10% FBS
and 100 U/mL penicillin and 100 μg/mL streptomycin. They were
maintained at 37 °C in a humidified atmosphere with 5% CO2. The cells
were split constantly after a confluent monolayer has been formed. To
split cells, the plate was washed briefly with phosphate-buffered saline
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(methanesul-
fonyl)-1,4,10,13-tetraoxa-7-azatridecane (35). A round-bottom
flask was charged with compound 14a (80 mg, 0.13 mmol),
methanesulfonyl chloride (30 mg, 0.26 mmol), and pyridine (10 mL).
The reaction mixture was stirred at room temperature for 14 h. When
TLC indicated complete consumption of starting material, the reaction
mixture was poured into a separating funnel containing 1 M hydrochloric
acid solution. The mixture was continuously extracted with CH2Cl2. The
combined organic layers were dried over MgSO4, filtered, and evaporated
to give a crude reaction mixture, which was subjected to purification by
flash column chromatography on silica gel with 10−20% acetone in
CH2Cl2 as eluent to furnish the titled compound 35 as a pale brown oil
1
(56 mg, 60%): H NMR (CDCl3) δ 2.93 (s, 3H), 3.53 (t, J = 5.2 Hz,
4H), 3.72 (t, J = 4.8 Hz, 4H), 3.81 (t, J = 4.8 Hz, 4H), 4.13 (t, J = 4.8 Hz,
4H), 6.68 (s, 2H), 6.97 (d, J = 8.8 Hz, 4H), 7.35 (dd, J = 7.6, 7.6 Hz, 2H),
7.49 (d, J = 8.4 Hz, 2H), 7.63 (dd, J = 8.0, 8.4 Hz, 2H), 7.82 (d, J = 8.8
Hz, 4H), 8.19 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3) δ 38.8, 47.5, 67.4,
69.3, 70.0, 106.1, 114.8, 117.9, 123.8, 124.2, 125.1, 125.5, 128.0, 133.6,
156.1, 161.4, 163.1, 178.2; LRMS (ESI) m/z 712 (M+ + H, 62), 734 (M+
+ Na, 90); HRMS (ESI) calcd for C39H38NO10S (M+ + H) 712.2216,
found 712.2224.
1,13-Bis[4′-(4H-chromen-4-on-2-yl)phenyl]-N-(4-methylben-
zenesulfonyl)-1,4,10,13-tetraoxa-7-azatridecane (36). A round-
bottom flask was charged with compound 14a (80 mg, 0.13 mmol),
p-toluenesulfonyl chloride (40 mg, 0.21 mmol), and pyridine (10 mL).
The reaction mixture was stirred at room temperature for 14 h. When
TLC indicated complete consumption of starting material, the reaction
mixture was poured into a separating funnel containing 1 M hydro-
chloric acid solution. The mixture was continuously extracted with
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dx.doi.org/10.1021/jm201121b | J. Med. Chem. 2012, 55, 1999−2014

Journal of Medicinal Chemistry
Article
(PBS), treated with 0.05% trypsin/EDTA, and harvested by centrifuga-
tion.
Compound 18 Accumulation. A quantity of 7.5 × 106 LCC6 and
LCC6MDR cells suspended in 5 mL of DMEM was incubated with 10
or 100 μM 18 for 120 min at 37 °C. After incubation, the cells were
washed and resuspended with 500 μL of lysis buffer (acetonitrile with
20% formic acid). The cells were lysed by repeated freeze−thaw cycles.
The level of 18 in the lysate was determined by high performance
liquid chromatography (HPLC). The HPLC system consisted of an
Agilent 1100 pump, UV−visible detector, and an automated sample
injector with a 100 μL loop. Chromatographic separation of 18 was
carried out in a 17 min run by Agilent Prep-sil column (440905-901,
4.6 mm i.d. × 250 mm, 5 μm). The elution profile consisted of
hexane−methanol−ethyl acetate (45:15:40, v/v/v) from 0 to 7 min.
Then a gradient elution of hexane−methanol−ethyl acetate (45:15:40,
v/v/v) to hexane−methanol−ethyl acetate (10:80:10, v/v/v) was applied
from 7 to 10 min following, and the condition was gradually changed back
to hexane−methanol−ethyl acetate (45:15:40, v/v/v) from 10 to 11 min
to equilibrate the column for the next injection. The flow rate was
1.0 mL/min. Eluent was recorded at 315 nm with reference to 450 nm.
Then 500 μL of cell lysate was alkalized by 100 μL of sodium hydroxide
(15M) and dried in an 80 °C dry bath. The dried samples were recon-
stituted with 100 μL of chloroform and immediately passed through a
13 mm × 0.22 μm nylon syringe filter (Chelleson Scientific Instruments
Company, Hong Kong, China). About 20 μL of the sample was injected
into the HPLC-DAD for quantitative analysis. Standard stock solution of
18 was prepared at 0.5 mg/mL by accurately weighing the required
amount and dissolving in chloroform. The calibration curve was con-
structed by serial dilution of 18 in chloroform from 25 to 0.25 μg/mL. All
prepared solutions were immediately injected into the HPLC instrument
for calibration curve construction.
Cell Proliferation Assay. Six-thousand cells of LCC6 or
LCC6MDR and paclitaxel were mixed with or without 0.5 and
1.0 μM modulator to a final volume of 200 μL in each well of 96-well
plates. Four-thousand cells of 2008/P or 2008/MRP1 and DOX were
co-incubated with or without 0.5 μM 18 or 8 to a final volume of
200 μL. Seven-thousand-five-hundred cells of HEK293/pcDNA3.1 or
HEK293/R2 and topotecan were co-incubated with or without 0.5 μM
18 or 9 to a final volume of 200 μL. The plates were then incubated
for 5 days at 37 °C. After 5 days, the % of survival or viability was
determined by MTS according to procedures reported previously.36
These results were represented as the mean standard error of mean.
IC50 values were calculated from the dose−response curves of MTS
assays (Prism 4.0).
Cytotoxicity Assay. The cytotoxicity assay of flavonoid dimers
was performed against LCC6, LCC6MDR, and L929 cell lines in order
to determine the relative toxicity. The cells were grown in
supplemented DMEM in an atmosphere of 95% air with 5% CO2 at
37 °C. In brief, LCC6, LCC6MDR, and L929 cells were seeded into
96-well flat bottom microtiter plate at 1 × 104 cells per well in a final
volume of 100 μL of medium, respectively. A graded dose of flavonoid
dimers was added into the wells. The plate was incubated at 37 °C for
72 h in an atmosphere of 5% CO2 in air. The % of survivors was deter-
mined as described previously.
DOX Accumulation. DOX accumulation assay was carried out
according to the reported procedures.36 Briefly, 1 × 106 cells of LCC6
and LCC6MDR were added in an Eppendorf and incubated with
20 μM DOX and different concentrations of 18 or 1 for 150 min at
37 °C. A 0.4% of DMSO was used as a negative control. After incuba-
tion, the cells were washed and lysed with lysis buffer (0.75 M HCl,
0.2% Triton-X100 in isopropanol). The fluorescence level of DOX in
the lysate was determined by fluorescence spectrophotometer (BMG
Technologies) using an excitation and an emission wavelength pair
of 460 and 610 nm.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra, 13C NMR spectra of all compounds, and
HPLC chromatogram of compound 18. This material is
available free of charge via the Internet at http://pubs.acs.org.
For the accumulation test, the log of the agonist versus response
variable-slope model was used to determine the Hill coefficient by
nonlinear regression to the equation Y = bottom + (top − bottom)/
AUTHOR INFORMATION
■
1 + 10(logEC −X)Hill coefficient, where “bottom” is the baseline and “top” is
50
Corresponding Author
the maximum effect, EC50 is the dose giving half the maximum effect,
and X is the modulator concentration (log). The cells were incubated
with 20 μM DOX and various dosages of 18 or 1 (40, 30, 20, 10, 8,
6, 4, 2, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005 μM).
After incubation, the intracellular DOX level was measured by cell
lysis followed by fluorescence spectrophotometry as described
previously.
*For L.M.C.C.: phone, (852)-34008662; fax, (852)-23649932;
e-mail, bclchow@polyu.edu.hk. For T.H.C.: phone, (852)-
34008670; fax, (852)-23649932; e-mail, bcchanth@polyu.edu.hk.
Author Contributions
∥These two authors contributed equally to this work.
Kinetic Characterization for P-gp Inhibition. Kinetic parame-
ters for P-gp inhibition by modulators were determined by incubating
the 1 × 106 LCC6MDR cells with various concentrations of DOX (1.5,
3, 4.4, 13.3, 20, 30, and 40 μM) in the presence of 18 (0, 0.006, 0.025,
0.05, 0.075, and 0.1 μM) or 1 (0, 0.06, 0.25, 0.5, 0.75, and 1 μM) for
150 min at 37 °C. The cell lysis and fluorescence intensity measure-
ments were carried out as described previously. The relationship
between DOX and modulators for P-gp inhibition was analyzed by
Lineweaver−Burk and Dixon plots as reported previously.36,49
P-gp-ATPase Assay. P-gp-ATPase activity was measured with the
P-gp-Glo assay system (Promega) with human P-gp membrane.34 The
assay relies on the ATP dependence of the light-generating reaction of
firefly luciferase. First, about 25 μg of human P-gp membrane fraction
was incubated with (1) 100 μM sodium vanadate and 0.5% DMSO,
(2) 0.5% DMSO, (3) 200 μM 1, and (4) 200 μM 18, respectively.
Second, the reaction was initiated by addition of 5 mM MgATP and
then the plate was incubated at 37 °C for 1 h. Third, after incubation,
the remaining unmetabolized ATP was determined as a luciferase-
generated luminescence signal by addition of ATP detection reagent.
Fourth, the plate was further incubated at room temperature for 20
min for signal stabilization and then the luminescence signal was
measured with GloMax-20/20 luminometer (Promega). ATPase
activity was presented as a drop in luminescence of samples compared
to that treated with sodium vanadate.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for funding support from the Hong Kong
Polytechnic University (Grants G-YE45, G-U383, and 1-BB8S)
and the Hong Kong Research Grants Council (Grants B-Q423,
B-Q762, and B-Q16G).
ABBREVIATIONS USED
■
MDR, multidrug resistance; P-gp, P-glycoprotein; ABC, ATP
binding cassette; MRP1, multidrug resistance protein 1; BCRP,
breast cancer resistance protein; PEG, polyethylene glycol; RF,
relative fold; DOX, doxorubicin
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