Anion Transporters Based on Noncovalent Balance including Anion-π, Hydrogen, and Halogen Bonding

Article abstract of DOI:10.1021/acs.joc.9b00561

Anion transmembrane transport mediated by novel noncovalent interactions is of central interest in supramolecular chemistry. In this work, a series of oxacalix[2]arene[2]triazine-derived transporters 1 and 2 bearing anion-π-, hydrogen-, and halogen-bondin

Full text of DOI:10.1021/acs.joc.9b00561

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Article
Anion Transporters Based on Noncovalent Balance
Including Anion-#, Hydrogen and Halogen Bonding
Wen-Long Huang, Xu-Dong Wang, Sen Li, Rui Zhang, Yu-
Fei Ao, Jun Tang, Qi-Qiang Wang, and De-Xian Wang
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 17 Jun 2019
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Anion Transporters Based on Noncovalent Balance Including
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Anion-, Hydrogen and Halogen Bonding
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Wen-Long Huang,a,b,# Xu-Dong Wang,a,# Sen Li,a Rui Zhang,c Yu-Fei Ao,a Jun Tang,c
Qi-Qiang Wang,a,b,* and De-Xian Wanga,b,*
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a Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of
Molecular Recognition and Function, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190 (China)
b University of Chinese Academy of Sciences, Beijing 100049 (China)
c State Key Laboratory of Agrobiotechnology and College of Veterinary Medicine,
China Agricultural University, Beijing, 100193, China
# These authors contribute equally
qiqiangw@iccas.ac.cn dxwang@iccas.ac.cn
Abstract: Anion transmembrane transport mediated by novel noncovalent
interactions is of central interest in supramolecular chemistry. In this work, a series of
oxacalix[2]arene[2]triazine-derived transporters 1 and 2 bearing anion-, hydrogen
and halogen bonding sites in rational proximity were designed and synthesized by a
one-pot strategy starting from gallic acid ester derivatives and mono- or di-halogen
1
substituted triazines. H NMR titrations demonstrated efficient binding of 1 and 2
towards Cl- and Br- in solution, giving association constants in the range of 102–104
M-1. Cooperation of anion-, hydrogen and halogen bonding was revealed as driving
force for anion binding by single crystal structures of two complexes and DFT
calculations. Fluorescence assays indicated compounds 1 are efficient chloride
transporters with effective concentrations (EC50) falling in the range of 3.1 to 7.4 M
and following an order of 1a > 1b > 1c > 1d. The contribution of halogen bonding
and cooperative noncovalent bonds to ion transport was then discussed. Significantly,
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transporters 1 exhibits high anticancer activity. In the presence of 1 and KCl (60 mM),
cell survival of HCT116 reduces to 11.9 – 24.9% with IC50 values in the range of 52.3
– 66.4 M.
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Introduction
Ion transport across cell membrane represents one of the most essential processes in
many living cells. Ion selectivity is the fundamental property of ion-transport proteins,
which is achieved by specific noncovalent interactions and stereo-configuration of the
binding sites.1 Inspired by the mysterious function and mechanism of transmembrane
proteins, design of synthetic ion transporters based on noncovalent interactions has
been one of the most attractive research fields in supramolecular chemistry.2 While a
large amount of artificial cation transporters are known, artificial systems for anion
transport are relatively rare.2 This most probably stemmed from the challenge of
anion recognition due to the intrinsic properties of anions such as large size, low
electric density and easy polarization, etc. Anion transport has been achieved
generally by hydrogen bond-based transporters such as prodigiosin mimics,3 peptide
derivatives,4 steroidal scaffolds with (thio)urea sidechains,2e,5 artificial -barrels,2b,6
and macrocyclic scaffolds bearing hydrogen bond donors.7 On the other hand, recent
years have witnessed a vast development of halogen bonding and anion-
interactions.8 Both noncovalent bonds as alternatives of hydrogen bonding show
remarkable capability in anion binding and anion recognition, however, halogen
bond- and anion--based anion channel and transporters are surprisingly limited.
Matile and coworkers reported the anion- oligonaphthalenediimide rods for selective
anion transport.9 Macrocyclic or semi-cage artificial anion transporters comprising
electron-deficient aromatic rings were demonstrated by several groups including us.10
Self-assembled chloride carriers or channels exploiting halogen bonding as driving
force were reported independently by Matile’s10b,11 and Zeng’s groups.12 These
reports, though still very few, have confirmed the desirable nature of halogen bonding
and anion- interactions for design of anion transport systems.13 It is worth addressing
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that the development of novel noncovalent interactions could in return help us get
deep understanding of the mechanism of biological ion transport. For example, the
function of anion- interaction in voltage-dependent response prestin was recently
realized by He and Beisel et al. They applied a Gltph-based model and demonstrated
the aromatic residues bind intracellular anions through anion- interactions; such
weak interactions are sensitive to voltage and mechanical stimulation and hence can
explain the voltage-dependent property of prestin.14 Therefore it is attractive and
important to explore novel noncovalent interactions in design of artificial ion
transport system, and to elucidate the contribution and probe the balance of various
noncovalent bonds.
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Dichloro-substituted oxacalix[2]arene[2]triazine is a typical member of
heteracalixaromatics.15 This preferent host adopts 1,3-alternate conformation in which
two triazine rings form an electron-deficient V-shape cleft and shows powerful
binding ability to anions through anion- interactions.16 The convergent cavity and
convenient functionalization of dichloro-substituted oxacalix[2]arene[2]triaizne
endow a good molecular platform to achieve cooperativity of various noncovalent
interactions. Previously, through introducing hydrogen bond donors on the lower rim
of oxacalix[2]arene[2]triazine derivatives, we have shown cooperative anion- and
hydrogen bonding interactions can mediate chloride transmembrane transport.10a To
manipulate a balance involving more noncovalent interactions, we decided to further
introduce halogen bond sites on the upper rim of oxacalix[2]arene[2]triazines to creat
a convergent proximity of anion- pocket with hydrogen and halogen bonding sites
(Figure 1). Herein we reported the design and synthesis of such new transporters 1
and 2 and their anion transport studies. The contribution and balance of different
noncovalent bonds in mediating the ion transport was highlighted.
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X
X
Y
Y
Y
X
O
Y
X
HN
O
NH
O
O
O
O
N
N
N
N
N
N
N
N
OH
O
OH
O
O
N
O
O
N
O
N
O
N
O
O
O
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COOCH3
X = H, Y = Br
X = Y = Br
X = H, Y = I
X = Y = I
COOCH3
H3COOC
H3COOC
2a
2b
2c
2d
X = H, Y = Br
X = Y = Br
X = H, Y = I
X = Y = I
1a
1b
1c
1d
Figure 1. Structures of oxacalix[2]arene[2]triazine-based transporters 1 and 2.
Results and discussion
Design. Oxacalix[2]arene[2]triaiznes are a good macrocyclic platform suitable
for organizing multiple noncovalent interactions in a cooperative manner. In order to
manipulate a balance between anion-, hydrogen and halogen bonding on the
macrocyclic scaffold, we decided to incorporate hydrogen bonding sites in the lower
rim of benzene rings, and further introduce halogen bonding sites on the upper rim of
triazine rings. The hydrogen bonding sites can choose hydroxyl or amide groups, and
halogen bonding sites can take bromo or iodo substituted phenyl groups. Considering
the macrocyclic geometry and synthetic feasibility, we designed two types of
transporters, 1 and 2. To evaluate whether the V-shape anion- cavity and the
hydrogen and halogen bonding sites are in good convergent proximity and can
cooperatively bind anions, e.g. chloride and bromide, we performed DFT calculations
on binding of 1b with Cl- and Br- on the base of B3LYP 6-31G*. To our delight the
optimized structures for both the anion complexes showed a well cooperative binding
motif in which the anion sits above the electron-deficient triazine plane and
simultaneously interacts with the two hydroxyl groups and one bromo site from the
pendant phenyl group (Figure S47).
Synthesis. In order to quickly access the ransporters 1 and 2, the straightforward
and convenient one-pot strategy was applied (Scheme 1). The dichloro-substituted
triazine monomers 3 were facilely prepared through nucleophilic substitution of
cyanuric chloride with a series of phenol derivatives containing bromo or iodo
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halogen-bonding groups. One-pot reactions between the gallic acid ester derivatized,
benzyl protecting diphenol monomers 4 and the triazine monomers 3 in the presence
of K2CO3 as acid scavenger in refluxed acetonitrile readily afforded the macrocyclic
compounds 5 in 41-65% yields. The following deprotection of 5 with an excess
amount of AlCl3 in dry toluene proceeded smoothly at ambient temperature and gave
transporters 1 in 68-82% yields. For synthesis of transporters 2, gallic acid ester
derived diphenol monomer 9 bearing amide substituent on 4-hydroxy position was
firstly prepared by two simple substitution and deprotection steps from 6. The one-pot
reactions between the two monomers 9 and 3 gave transporters 2 in moderate yields.
The successful synthesis of 1 and 2 represents a rare example on facile one-pot
preparation of functionalized oxacalix[2]arene[2]triazines with considerable yields.
For synthesis of the macocyclic analogue 12 without halogen-bond donor sites,
the dichloro-substituted macrocyclic precursor 10 was firstly reacted with two
equivalents of phenol in the presence of K2CO3 under reflux to give the product 11 in
51% yield (Scheme 2). The following debenzylation with AlCl3 in toluene provided
the desired macrocycle 12 in 76% yield. All the compounds were fully characterized
with spectroscopic and elemental analyses.
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COOCH3
COOCH3
O
OBn
N
OBn
HO
OH
Cl
N
Cl
N
O
N
O
X
X
Y
C
OBn
4
X
N
N
N
N
N
N
N
N
OH
O
X
Y
O
K2CO3
Cl
Cl
Cl
O
K2CO3, CH3CN
reflux
acetone, 0 o
O
Y
Y
3a
3b
3c
3d
X = H, Y = Br 54%
X = Y = Br 47%
X = H, Y = I 55%
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COOCH3
X = Y = I
53%
5a
X = H, Y = Br 63%
5b
5c
5d
X = Y = Br
X = H, Y = I 57%
X = Y = I 41%
65%
COOCH3
O
N
O
X
N
O
N
O
AlCl3
toluene, rt
1a
X = H, Y = Br 74%
N
N
OH
OH
1b
1c
1d
X = Y = Br
X = H, Y = I 82%
X = Y = I 68%
70%
O
X
Y
O
N
Y
COOCH3
COOCH3
COOCH3
O
COOCH3
OBn
Br
N
H
7
H2, Pd/C
HO
BnO
OH
OBn
O
CH3OH, rt
66%
K2CO3, DMF, rt
70%
BnO
O
O
OH
NHPh
O
NHPh
6
9
8
COOCH3
Cl
O
O
O
X
N
X
2a
2b
2c
2d
X = H, Y = Br 20%
X = Y = Br 62%
X = H, Y = I 17%
X = Y = I 40%
N
N
9
N
O
N
Y
PhHN
O
NHPh
N
N
X
Y
O
O
K2CO3, CH3CN
reflux
Cl
N
O
N
O
Y
3a-3d
O
O
COOCH3
Scheme 1. Synthesis of macrocyclic transporters 1 and 2.
COOCH3
COOCH3
O
OBn
N
OBn
O
N
O
O
N
O
O
N
O
N
N
N
N
N
N
N
N
OBn
OBn
OH
Cl
O
Cl
O
K2CO3, CH3CN
reflux
O
51%
COOCH3
COOCH3
10
11
COOCH3
O
N
O
N
O
AlCl3
N
N
OH
OH
toluene, rt
O
N
O
76%
N
O
COOCH3
12
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Scheme 2. Synthesis of macrocycle 12 without halogen-bond donor sites.
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Anion Binding Studies. The affinity of 1 and 2 towards Cl- and Br- (as Bu4N+
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1
salts) was assessed by H NMR titrations in d6-acetone (Figures S3-S28). From the
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NMR spectra illustrated in Figures S3-S6, treatment of 1 with chloride caused
significant downfield shifts of the hydroxyl proton signals, along with slight upfield
shifts of the proton signals on the halogen substituted benzene. The downfield shift of
hydroxyl proton signal suggests the consistent inclusion of chloride within the
electron-deficient V-shape cavity, where the hydroxyl protons co-participate in the
anion binding through hydrogen bonding.17 The upfield shifts of the
halogen-substituted benzene proton signals can be attributed to the formation of
halogen bonding. Chloride binding with 1 is therefore achieved by a combination of
anion-, hydrogen and halogen bonding. Interaction of 1 with bromide resulted in
different chemical shift changes. Besides the general upfield shifts for the
halogen-substituted benzene proton signals, the hydroxyl proton signals, however,
gradually moved to upfield upon the addition of bromide, which seems strange at first
glance. This unusual upfield shift of the hydroxyl signals could reflect a subtle
balance between the hydrogen-bonding induction effect and aromatic ring shielding
effect. As suggested by the calculation (Figure S47), upon binding the hydroxyl
proton was pulled above the triazine ring and shielded by the aromatic plane. In case
of bromide, the shielding effect caused upfield shift could be more predominant than
hydrogen-bonding induced downfield shift due to its less hydrogen bonding ability
than chloride. In contrast, hosts 2 showed similar response to both halides. The
significant downfield shifts of amide proton signals and slight upfield shifts of
halogen-substituted aryl proton signals suggested consistent halide binding through
combination of hydrogen and halogen bonding. The strength of anion binding (Ka1:1
)
was calculated by fitting the titration data with HypNMR 2004 program. The 1:1
binding stoichiometry was further verified by a Job’s plot experiment (see Supporting
Information). As shown in Table 1, 1 and 2 show strong affinity to halides, giving
association constants in the range of 102–104 M-1, and overall stronger binding
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towards chloride than bromide. Moreover, for the two types of transporters, the
association constants nearly follow the same order of a < b < c < d. As the
halogen-bond donor ability follows an order of Cl < Br < I,8a the consistent order for
the binding affinity further confirms the contribution of halogen bonding.
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Table 1. Association Constants (Ka1:1, M-1) of 1, 2 with Bu4N+Cl- and Bu4N+Br-[a]
Bu4N+Cl-
Bu4N+Br-
1a
1b
1c
1d
2a
2b
2c
2d
(7.37 ± 0.32) × 103
(9.38 ± 0.05) × 103
(3.43 ± 0.46) × 104
(1.30 ± 0.18) × 104
(6.03 ± 0.06) × 103
(4.15 ± 0.27) × 103
(9.00 ± 0.11) × 103
(4.70 ± 1.39) × 104
(1.00 ± 0.06) × 103
(6.86 ± 0.11) × 102
(1.55 ± 0.19) × 103
(6.34 ± 0.18) × 102
(8.97 ± 0.05) × 102
(5.25 ± 0.08) × 102
(1.70 ± 0.14) × 103
(1.04 ± 1.37) × 104
[a]
1
Determined by H NMR titrations in d6-acetone. The association constant was
obtained by fitting the titration data with HypNMR 2004 program.
Crystallographic Studies. To probe the contribution of different types of
noncovalent bonds to anion binding from molecular level, single crystals of anion
complexes with halides were cultivated. Pleasantly, single crystals of high quality for
complexes [1bCl]- and [1cCl]- were obtained and their structures were determined.
As illustrated in Figures 2-3, in both complexes, chloride is included within the
electron-deficient cavity formed with triazines through multiple noncovalent
interactions including anion-, hydrogen and halogen bonding. The different
interaction details depending on the host structures are worth addressing. In case of
[1bCl]-, a hydrogen bonded Cl5-O14-Cl4 guest triplet is stabilized within the
V-shape host cavity. In particular, Cl5 locates above one triazine ring with a short
contact (dCl5-C20 = 3.204 Å), indicating the formation of anion- interaction.
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Moreover, it forms hydrogen bonding with one hydroxyl group (dCl5-O10 = 2.981 Å)
5
6
and halogen bonding with one bromine (Br3) on the adjacent macrocycle (dCl5-Br3
=
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3.468 Å). Notably, the distance between Cl5 and neibouring Br1 (dCl5-Br1 = 3.998 Å)
is considerably larger than the sum of their van der Waals radius, suggesting
negligible interaction. Such result is most probably due to that the strict directionality
of halogen bonding disfavors its intra-cavity cooperation with anion- and hydrogen
bonding. The multiple noncovalent interactions involved with chloride (Cl5) facilitate
intermolecular self-assembly to form an extened structure as shown in Figure 2B.
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Figure 2. Crystal structure of [1bCl]- complex. (A) Participation of noncovalent
interactions in chloride binding and (B) intermolecular self-assembly. Countercations
and nonessential hydrogen atoms are omitted for clarity. Ellipsoid probability is 25%.
In case of [1cCl]-, two types of capsule-like structures, namely, capsule 1 and
capsule 2 as shown in Figure 3A and 3B are observed. In capsule 1, two
iodine-substituted aryl groups adopt a syn orientation on the macrocyclic skeleton and
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two of such macrocycle molecules form a semi-cage cavity through intermolecular
halogen-bonding between the inward iodines (dI2A-I1A = 3.906 Å). Chloride (Cl1) is
included within the cavity through cooperative anion- (dCl1-plane = 3.086 Å) and
duplicate hydrogen bonds (dCl1-O6A = 2.943 Å, dCl1-O10A = 3.100 Å). Halogen bonding
doesn’t participate in the binding with chloride as judged from the long distance to the
adjacent I2A (dCl1-I2A = 4.645 Å). The countercation, Et4N+, is included in between the
two chlorides to compensate the electrostatic repulsion. In contrast, in capsule 2, the
two iodines (I1 and I2) are in anti orientation and no longer convergent. Besides the
similar, cooperative anion- and hydrogen bonding as observed in capsule 1, the
chloride (Cl2) also enjoys halogen bonding with I1. These noncovalent interactions
direct the formation of a more oblate capsular structure. Notably, the outward iodines
(I2) from two adjacent macrocycles form halogen bonding with a “free” chloride
(Cl3), leading to one-dimension self-assembly (Figure 3C).
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Figure 3. Crystal structure of [1cCl]- showing two types of capsular structures. (A)
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Capsule 1, (B) capsule 2, and (C) intermolecular self-assembly of capsule 2.
Nonessential countercations and hydrogen atoms are omitted for clarity. Ellipsoid
probability is 25%.
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Anion Transport Studies. Encouraged by the anion binding study, we then
performed a fluorescence assay to evaluate the anion transport activities of 1 and 2. In
this assay, large unilamellar vesicles composed of egg yolk phosphatidylcholine
(EYPC LUVs) were loaded with halide selective dye lucigenin as anion transport
indicator, then the target transmembrane anion gradient across the membrane was
applied. After the addition of transporters, the fluorescent intensity with time was
recorded. The fluorescence kinetics of chloride transport in the presence of 1 and 2 (5
M) are shown as Figure 4.
1a
1b
1c
1d
1.2
1.0
2a
2b
2c
2d
0.8
DMF
0.6
0.4
0.2
0.0
0
50
100
150
200
250
300
350
Time (s)
Figure 4. Evaluation of the chloride transport activity of transporters 1 and 2 by
lucigenin  EYPC-LUV fluorescence assays (concentration for each compound is 5
M).
Surprisingly, although the two series of host compounds bind chloride with
comparable affinity, only 1a-d give significant chloride transport activity. To
understand the contrasting transport behaviors of 1 and 2, we did Hill analyses of the
dose-response curve with NaCl as the extravesicular salt for transporters 1a-d. The
effective concentrations (EC50) and Hill coefficients (n) are compiled in Table 2 (for
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graph see Figures S30-S33). The EC50 values for chloride transport are in the range of
3.1 to 7.4 M, and suggest an order of transport activity 1d > 1c > 1b > 1a. Such
halogen-bond dependent order indicates the contribution of halogen bonding to
chloride transport. When we applied 1a-d to bromide transport assay, the transporters
gave same halogen-bond dependent activities, which further indicates the function of
halogen bonding. For contribution of other noncovalent bonds, we applied two
structural anlogues to investigate the structure-activity relationship. Removing of the
two lower-rim hydroxyl groups and also the ester groups (compound 13,18 Figure
S29) caused complete inactivation of chloride transport. Alternatively, removing of
the halo atoms (compound 12) showed comparable activity (Table 2, Figure S34) to
that of 1d, which on one hand highlights the contribution of hydroxyl groups, on the
other hand suggests the largely reduced thermodynamic chloride binding (Ka = 560
M-1) to 12 could increase the transport activity.19 The other explanation could be that
different “active” strutures are possibly responsible for the transport, e.g. a capsular
assembly for 1 and a simpler molecular structure for 12. Taken together, the ion
transport is probably mediated by a balance and cooperation of different noncovalent
bonds in the molecular backbone. Besides, the obtained Hill coefficients for chloride
and bromide are higher than 1 (n > 1), suggesting that halogen-bonded capsule (as
shown in Figure 3) may act as the active entity for anion transport. The negligible
transport activity of 2a-d once again suggests the importance of the closely attached
low-rim hydroxyl groups.
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Table 2. EC50 (μM, mol%) and Hill coefficients (n) of 1a-d with Various Salts.
LiCl
9.4±0.5
5.4
NaCl
7.4±0.5 4.6±0.2 4.0±0.4 4.8±0.2 10.3±1.7
4.3 2.7 1.7 2.8 6.0
KCl
RbCl
CsCl
NaBr
EC50 (μM)
EC50 (mol%)
n
1a
1b
2.8±0.6
6.6±0.2
3.8
3.2±0.7 7.8±2.0 4.6±2.0 6.5±2.8 3.0±1.7
5.5±0.1 5.1±0.4 4.0±0.1 4.4±0.2 7.2±0.6
EC50 (μM)
EC50 (mol%)
3.2
3.0
2.3
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n
3.3±0.4
4.1±0.4 4.2±2.2 4.7±0.4 4.5±0.9 3.2±0.9
4.0±0.2 3.9±0.1 3.5±0.1 2.9±0.1 7.0±1.1
EC50 (μM)
EC50 (mol%)
n
5.4±0.7
3.1
1c
1d
12
2.3
2.3
2.0
1.7
4.1
3.3±1.1
3.2±0.1
1.9
3.2±0.6 2.9±0.9 4.4±0.6 4.5±0.7 2.4±0.9
3.1±0.1 2.8±0.1 2.8±0.2 2.7±0.1 2.8±0.1
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EC50 (μM)
EC50 (mol%)
n
1.8
1.6
1.6
1.6
1.6
3.3±0.3
2.2±0.2 3.0±0.3 3.5±0.7 3.0±0.2 3.4±0.3
2.2±0.7
EC50 (μM)
EC50 (mol%)
n
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-
1.3±0.7
To probe the transport mechanism, fluorescence assay with LUVs containing
carboxyfluorescein (CF) was performed. Upon the addition of transporters 1a-d, no
fluorescence enhancement was observed, indicating the transporters do not induce the
membrane defects (Figure S35). We then set up assays applying vesicles prepared
with dipalmitoyl phosphatidylcholine (DPPC), a lipid showing a gel to liquid
crystalline transition phase at 41 C. Transport assay at 25 C gave dramatically
reduced transport activity due to the decrease of membrane fluidity, suggesting that
the ion transport process is governed by a carrier mechanism (Figure S37). To
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investigate whether the transport is through a Cl-/NO3 antiport process, we prepared
the vesicles containing lucigenin and sodium chloride. Nitrate and the control anion
sulfate were added to the extravesicular buffer solution, respectively. In both cases the
intensity of quenched lucigenin was recovered with the release of chloride to the
-
extravesicular buffer, showing independence of chloride efflux on external NO3 or
SO42- (Figure S36). As sulfate is an extremely hydrophilic anion and it is normally
impossible to be transported from aqueous solution to the lipid bilayer membrane, the
results thus indicate the less possibility of a Cl-/NO3 antiport exchange. The influence
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of the external cations (alkali metal) on the transport activity was further investigated.
As illustrated in Table 2, the transport activities (EC50s) of 1a-d show cation
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dependence. For different cations, the activities follow an overall sequence of Li+ <
Na+ < K+  Rb+  Cs+. These outcomes suggest the ion transport is most probably
through an X-/M+ ion-pair process. To further shed light on the transport mechanism,
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i.e. Cl-/M+ or Cl-/NO3 , we performed HPTS/FCCP experiment with 1d chosen as a
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typical example. According to the results, the activity of 1d in the presence of FCCP
is only a sum of 1d and FCCP alone, and no additional effect was observed (Figure
S38). Besides, we also applied the potassium transporter valinomycin to the lucigenin
 EYPC-LUV system. The coexistence of 1d and valinomycin showed a similar
activity in comparison with 1d alone (valinomycin alone did not show any significant
activity) (Figure S39). Hence both the experiments further supported the Cl-/M+
mechanism. The ion pair binding mode could be similar to that observed in crystal
structure, i.e. X- is bound through the multiple noncovalent interactions, where M+ as
countercation sits around and interacts with hydroxyl oxygen atom through ion-dipole
interaction.
Evaluation of Anticancer Activities. The efficient transport activity
demonstrated by transporters 1 encouraged us to examine their possible application as
anticancer chemotherapy agents.12 The anticancer activity of transporters was
evaluated using the colorectal carcinoma cell line HCT116 which were cultured in
Dulbecco’s Modified Eagle Medium in the presence of KCl with different
concentrations of transporters ranging from 0 to 200 M (Figure 5). For comparison,
the anticancer activities of 2c, 2d and compound 13 were also investigated. Cell
viabilities determined after culturing the cells for 1 day at 37 C with 5% CO2 in the
presence of transporters 1, show that HCT116 cells are sensitive to the drugs in a
concentration dependent manner. As shown in Figure 5, in the presence of 60 mM
KCl, addition of transporters 1 ([1] = 200 M) lead to 11.9 (1a), 24.9 (1b), 14.9 (1c)
and 16.0% (1d) cell survival, indicating their efficient ability to inhibit tumor cell
growth with 1a being the most potent one. The IC50 values calculated are 52.3 M for
1a, 58.9 M for 1b, 66.4 M for 1c and 54.8 M for 1d. Not surprisingly, the control
compounds 2c, 2d and 13 did not show significant effect on the cell growth,
suggesting the correlation between transport activity and anticancer activity. We
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further determined the cell viabilities at different concentrations of KCl with various
concentrations of transporters. All four transporters (1a, 1b, 1c, 1d) show good
anticancer activities while the control molecules did not (Figures S40-S46).
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1a
1b
[KCl]=60 mM
1c
1d
2c
2d
13
100
80
60
40
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0
50
100
150
200
Concentration(uM)
Figure 5. Viabilities of HCT116 cells in the presence of various concentrations of
transporters or control compounds at [KCl] = 60 mM.
Conclusions
In conclusion, we have designed oxacalix[2]arene[2]triazine-derived ion transporters
1 and 2 by incorporating multiple noncovalent bonds in close proximity. The
manipulation of noncovalent balance was exemplified by anion recognition study in
solution and solid state. Both compounds showed strong halide affinity through
combination of anion-, hydrogen and halogen bonding. Fluorescence assays for
transmembrane activity distinguished compounds 1 as efficient transporters.
Systematic study indicated the ion transport was mediated with a balance of multiple
noncovalent bonds including halogen bonding, hydrogen bonding and anion-
interactions. Transporters 1 were revealed to show efficient inhibit of HCT116 cell,
which promise their future as potential anticancer chemotherapy agents.
EXPERIMENTAL SECTION
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General Information. Reagents for synthesis and analysis were purchased from J&K
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or Sigma-Aldrich. Egg yolk phosphatidylcholine (EYPC) and a Mini-Extruder used
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for vesicle preparation were purchased from Avanti Polar Lipids. 1H and C NMR
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spectra were recorded on 300, 400 or 500 MHz spectrometers. Chemical shifts are
reported in ppm versus tetramethylsilane with either tetramethylsilane or the residual
solvent resonance used as an internal standard. Abbreviations are used in the
description of NMR data as follows: chemical shift (, ppm), multiplicity (s = singlet,
d = doublet, t = triplet, dd = doublet of doublets, m = multiplet), coupling constant (J,
Hz). Melting points are uncorrected. Infrared spectra were recorded on Nicolet-6700
FT-IR spectrometer. Mass spectra were obtained on BIFLEX Ⅲ, Bruker Inc.
(MALDI-TOF) and Bruker APEX-2 (HRMS). Elemental analysis was recorded on
Thermo Quest CE Instruments flash EA 1112 analyser. Single crystal X-ray
diffraction data were collected on a MM007HF Saturn724+ diffractometer using
MoK/α radiation (λ=0.71073 Å or 0.710747 Å) at a temperature of 173 K. All
anhydrous solvents were dried according to standard procedures prior to use. All other
major chemicals were obtained from commercial sources and used without further
purification.
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Synthesis Procedures.
General procedure for synthesis of 3. In an ice bath, anhydrous potassium
carbonate powder (20 mmol), cyanuric chloride (20 mmol) and acetone (100 mL)
were mixed in a flask. The solution of the corresponding phenol derivative (20 mmol)
in acetone (100 mL) was then added dropwise within 2 h. The resulting mixture was
stirred for 1 h in ice bath condition. After filtration, the filtrate was concentrated and
chromatographed on a silica gel column (100-200 mesh). The obtained solid was
recrystallized to give pure compound 3 as a white solid.
2,4-dichloro-6-(2-bromophenoxyl)-1,3,5-triazine (3a). Chromatography with a
mixture of petroleum ether and acetone (35/1, v/v) and recrystallized from hexane.
54% yield. mp 91-92 oC; 1H NMR (CDCl3/300 MHz) δ 7.68 (dd, J = 1.1, 7.6 Hz, 1H),
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7.42 (td, J = 1.4, 7.7 Hz, 1H), 7.27-7.21 (m, 2H); C{1H} NMR (CDCl3/75 MHz) δ
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173.4, 170.8, 148.6, 134.0, 129.2, 128.6, 123.1, 115.8; IR (KBr) ν 1521, 1403, 1200
cm-1; MS (EI-MS) m/z (%): 240 [M-Br]+ (100), 242 [M-Br]+ (50), 244 [M-Br]+ (10).
Anal. Calcd. for C9H4BrCl2N3O: C, 33.68; H, 1.26; N, 13.09. Found: C, 33.72; H,
1.26; N, 12.98.
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2,4-dichloro-6-(2,6-dibromophenoxyl)-1,3,5-triazine (3b). Chromatography
with a mixture of petroleum ether and acetone (30/1, v/v) and recrystallized from
hexane. 47% yield. mp 136-137 oC; 1H NMR (CDCl3/300 MHz) δ 7.63 (d, J = 8.1 Hz,
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2H), 7.12 (t, J = 7.8, 8.1 Hz, 1H); C{1H} NMR (CDCl3/75 MHz) δ 173.5, 170.0,
146.2, 133.0, 129.2, 117.2; IR (KBr) ν 1521, 1394, 1211 cm-1; MS (EI-MS) m/z (%):
318 [M-Br]+ (50), 320 [M-Br]+ (100), 322 [M-Br]+ (35), 324 [M-Br]+ (5). Anal. Calcd.
for C9H3Br2Cl2N3O: C, 27.03; H, 0.76; N, 10.51. Found: C, 27.10; H, 0.80; N, 10.57.
2,4-dichloro-6-(2-iodophenoxyl)-1,3,5-triazine (3c). Chromatography with a
mixture of petroleum ether and acetone (30/1, v/v) and recrystallized from hexane and
acetone. 55% yield. mp 97-98 oC; 1H NMR (CDCl3/300 MHz) δ 7.90 (dd, J = 1.2, 6.0
Hz, 1H), 7.45 (td, J = 1.2, 6.0 Hz, 1H), 7.18 (dd, J = 1.2, 6.0 Hz, 1H), 7.09 (td, J =
1.2, 6.0 Hz, 1H); 13C{1H} NMR (CDCl3/75 MHz) δ 173.3, 170.7, 151.5, 140.1,
130.1, 128.8, 122.5, 89.4; IR (KBr) ν 1527, 1402, 1193 cm-1; MS (EI-MS) m/z (%):
240 [M-I]+ (100), 242 [M-I]+ (50), 244 [M-I]+ (10). Anal. Calcd. for C9H4Cl2IN3O: C,
29.38; H, 1.10; N, 11.42. Found: C, 29.31; H, 1.16; N, 11.47.
2,4-dichloro-6-(2,6-diiodophenoxyl)-1,3,5-triazine (3d). Chromatography with
a mixture of petroleum ether and acetone (30/1, v/v) and recrystallized from hexane
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and acetone. 53% yield. mp 150-151 C; H NMR (CDCl3/300 MHz) δ 7.86 (d, J =
7.9 Hz, 2H), 6.80 (t, J = 7.9 Hz, 1H); 13C{1H} NMR (CDCl3/75 MHz) δ 173.5, 169.9,
151.3, 140.1, 130.1, 89.6; IR (KBr) ν 1523, 1394, 1206 cm-1; MS (CI-MS) m/z (%):
365.8 [M-I]+ (100), 367.8 [M-I]+ (67), 369.8 [M-I]+ (15). Anal. Calcd. for
C9H3Cl2I2N3O: C, 21.89; H, 0.61; N, 8.51. Found: C, 21.94; H, 0.78; N, 8.54.
General procedure for synthesis of 5. Fine anhydrous potassium carbonate
powder (10 mmol) and acetonitrile (100 mL) were mixed in a flask and heated at
reflux. The solution of 4 (4 mmol) and 3 (4 mmol) in acetonitrile (100 mL) was then
added dropwise within 2 h. The resulting mixture was refluxed for another 10 min and
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cooled to room temperature. After filtration, the filtrate was concentrated and
chromatographed on a silica gel column (100-200 mesh) to give pure compound 5 as
a white solid.
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5a. Chromatography with a mixture of petroleum ether, chloroform and acetone
(10/2/1, v/v/v). 63% yield. mp 174-175 oC; 1H NMR (CDCl3/300 MHz) δ 7.65 (d, J =
8.1 Hz, 2H), 7.59 (s, 4H), 7.40 (t, J = 7.5 Hz, 2H), 7.23-7.06 (m, 14H), 4.88 (s, 4H),
3.85 (s, 6H); 13C{1H} NMR (CDCl3/75 MHz) δ 173.7, 173.1, 164.8, 149.0, 146.5,
144.8, 135.6, 133.6, 128.9, 128.3, 128.2, 127.9, 127.7, 125.7, 123.4, 122.0, 116.2,
75.3, 52.4; IR (KBr) ν 1729, 1568, 1368, 1211 cm-1; MS (MALDI-TOF) m/z (%):
1065.0 [M+Na]+ (20), 1067.0 [M+Na]+ (100), 1069.1 [M+Na]+ (40). Anal. Calcd. for
C48H32Br2N6O12: C, 55.19; H, 3.09; N, 8.05. Found: C, 55.04; H, 3.32; N, 7.92.
5b. Chromatography with a mixture of petroleum ether, chloroform and acetone
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(10/3/1, v/v/v). 65% yield. mp >300 C; H NMR (CDCl3/300 MHz) δ 7.61-7.29 (m,
8H), 7.18-7.06 (m, 12H), 4.86 (s, 4H), 3.86 (s, 6H); 13C{1H} NMR (CDCl3/75 MHz)
δ 173.2, 173.0, 165.0, 146.8, 146.5, 145.0, 135.6, 132.8, 128.7, 128.5, 128.3, 128.0,
125.9, 122.2, 117.8, 75.4, 52.6; IR (KBr) ν 1730, 1562, 1360, 1224 cm-1; MS
(MALDI-TOF) m/z (%): 1224.8 [M+Na]+ (100), 1226.8 [M+Na]+ (60). Anal. Calcd.
for C48H30Br4N6O12: C, 47.95; H, 2.51; N, 6.99. Found: C, 47.92; H, 2.59; N, 7.12.
5c. Chromatography with a mixture of petroleum ether, chloroform and acetone
(9/2/1, v/v/v). 57% yield. mp 224-225 oC; 1H NMR (CDCl3/300 MHz) δ 7.86 (dd, J =
1.5, 6.6 Hz, 2H), 7.59 (s, 4H), 7.41 (td, J = 1.5, 6.6Hz, 2H), 7.23-7.02 (m, 14H), 4.93
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(s, 4H), 3.86 (s, 6H); C{1H} NMR (CDCl3/75 MHz) δ 173.7, 173.2, 164.9, 152.1,
146.6, 145.0, 139.9, 135.8, 129.9, 128.5, 128.3, 128.2, 127.8, 125.8, 122.8, 122.1,
90.3,75.4, 52.6; IR (KBr) ν 1728, 1567, 1367, 1207 cm-1; MS (MALDI-TOF) m/z
(%): 1011.1 [M-I]+ (100), 1139.0 [M+H]+ (25), 1161.0 [M+Na]+ (35). Anal. Calcd.
for C48H32I2N6O12: C, 50.63; H, 2.83; N, 7.38. Found: C, 50.47; H, 2.94; N, 7.33.
5d. Chromatography with a mixture of petroleum ether, chloroform and acetone
(5/1/0.4, v/v/v). 41% yield. mp 282-283 oC; 1H NMR (CDCl3/300 MHz) δ 7.81 (d, J =
7.8 Hz, 4H), 7.64 (s, 4H), 7.18-7.08 (m, 10H), 6.74 (t, J = 7.9 Hz, 2H), 4.96 (s, 4H),
3.86 (s, 6H); 13C{1H} NMR (CDCl3/75 MHz) δ 173.2, 172.8, 164.8, 151.8, 146.5,
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144.9, 139.8, 135.9,129.6, 128.6, 128.1, 127.8, 125.9, 122.2, 90.3, 75.2, 52.6; IR
(KBr) ν 1731, 1571, 1356, 1216 cm-1; MS (MALDI-TOF) m/z (%): 1412.3 [M+Na]+
(100). Anal. Calcd. for C48H30I4N6O12: C, 41.46; H, 2.17; N, 6.04. Found: C, 41.48;
H, 2.22; N, 5.96.
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General procedure for synthesis of 1. At room temperature, 5 (0.25 mmol),
anhydrous aluminum chloride (7.5 mmol) and dried toluene (20 mL) were mixed in a
flask. The mixture was stirred for 5 h under argon gas and poured into ice water (20
mL), then extracted with ethyl acetate (2×20 mL). The organic phase was dried with
anhydrous sodium sulfate and filtered. After removal of organic solvent, the residue
was recrystallized with ethyl acetate to give pure compound 1 as a white solid.
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1a. 74% yield. mp 239-240 C; H NMR (d6-acetone/300 MHz) δ 7.79 (dd, J =
1.5, 8.0 Hz, 2H), 7.55 (td, J =1.5, 8.2 Hz, 2H), 7.48 (s, 4H), 7.45 (dd, J =1.6, 8.1 Hz,
2H), 7.33 (td, J =1.6, 8.1 Hz, 2H), 3.79 (s, 6H); 13C{1H} NMR (d6-acetone/75 MHz)
δ 174.8, 174.5, 165.4, 150.2, 147.2, 141.2, 134.5, 130.0, 129.0, 124.9, 122.7, 121.7,
116.5, 52.4; IR (KBr) ν 3164, 1731, 1568, 1366, 1213 cm-1; MS (ESI) m/z (%): 861.3
[M]- (80), 863.3 [M]- (100), 865.3 [M]- (75). HRMS (ESI): m/z calcd. for [M+H]+
C34H21Br2N6O12: 862.9579; found 862.9562.
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1b. 70% yield. mp 262-263 C; H NMR (d6-acetone/300 MHz) δ 7.81 (d, J =
8.1 Hz, 4H), 7.50 (s, 4H), 7.30 (t, J =8.1 Hz, 2H), 3.79 (s, 6H); 13C{1H} NMR
(d6-DMSO/75 MHz) δ 173.0, 172.1, 164.4, 146.4, 145.9, 140.1, 133.0, 129.5, 121.8,
119.2, 117.1, 51.9; IR (KBr) ν 3445, 1720, 1576, 1363, 1223 cm-1; MS
(MALDI-TOF) m/z (%): 1045.0 [M+Na]+ (100), 1047.0 [M+Na]+ (50). HRMS (ESI):
m/z calcd. for [M+H]+ C34H19Br4N6O12: 1018.7789. Found: 1018.7770.
1c. 82% yield. mp >300 oC; 1H NMR (d6-acetone/300 MHz) δ 8.10 (dd, J = 1.2,
6.6 Hz, 2H), 7.56 (td, J =1.2, 6.6 Hz, 2H), 7.48 (s, 4H), 7.39 (dd, J =1.2, 6.6 Hz, 2H),
7.18 (td, J =1.2, 6.6 Hz, 2H), 3.79 (s, 6H); 13C{1H} NMR (d6-DMSO/75 MHz) δ
173.1, 172.9, 164.4, 151.6, 146.6, 140.1, 139.4, 129.9, 128.3, 123.3, 121.7, 119.2,
90.8, 51.9; IR (KBr) ν 3396, 1724, 1570, 1367, 1209 cm-1; MS (ESI-MS) m/z (%):
957.3 [M]- (100). Anal. Calcd. for C34H20I2N6O12: C, 42.61; H, 2.10; N, 8.77. Found:
C, 42.73; H, 2.24; N, 8.95.
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1d. 68% yield. mp >300 C; H NMR (d6-acetone/300 MHz) δ 8.02 (d, J = 7.9
Hz, 4H), 7.49 (s, 4H), 6.93 (t, J =7.9 Hz, 2H), 3.80 (s, 6H); 13C{1H} NMR
(d6-acetone/75 MHz) δ 174.6, 174.0, 165.4, 153.0, 147.2, 141.5, 141.0, 130.7, 122.7,
121.8, 91.1, 52.4; IR (KBr) ν 3203, 1724, 1575, 1358, 1217 cm-1; MS (MALDI-TOF)
m/z (%): 1210.4 [M+H]+ (20), 1232.3 [M+Na]+ (100). HRMS (ESI): m/z calcd. for
[M+H]+ C34H19I4N6O12: 1210.7234. Found: 1210.7207.
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Synthesis
of
methyl
3,5-bis(benzyloxy)-4-(2-oxo-2-(phenylamino)ethoxy)benzoate (8). At room
temperature, anhydrous potassium carbonate powder (332 mg, 2.4 mmol), 6 (729 mg,
2 mmol) and DMF (10 mL) were added to a flask and stirred for 1 h. Then 7 (2.2
mmol, 471 mg) was added and the reaction mixture was stirred for another 0.5 h. To
the mixture was added water (20 mL), and 10% dilute hydrochloric acid to adjust pH
= 7. After extracted with ethyl acetate, the organic phase was dried with anhydrous
sodium sulfate and filtered. The filtrate was concentrated and chromatographed on a
silica gel column (100-200 mesh) with a mixture of petroleum ether and ethyl acetate
(6/1, v/v) as an eluent. The obtained solid was recrystallized with petroleum ether and
ethyl acetate to give pure compound 8 as a colorless solid (770 mg, 70%). 8: mp
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111-112 C; H NMR (d6-acetone/300 MHz) δ 9.26 (s, 1H), 7.60-7.57 (m, 4H), 7.52
(s, 2H), 7.43-7.30 (m, 8H), 7.19 (m, 2H), 7.03 (m, 1H), 5.32 (s, 4H), 4.68 (s, 2H),
3.87 (s, 3H); 13C{1H} NMR (d6-acetone/75 MHz) δ 167.7, 166.6, 152.4, 142.3, 138.9,
137.5, 129.5, 129.1, 128.9, 126.9, 124.6, 120.3, 109.4, 73.4, 72.1, 52.6; IR (KBr) ν
3365, 1716, 1692, 1110 cm-1; MS (EI-MS) m/z 406 [M-Bn]+ (100%), 497 [M]+ (9).
Anal. Calcd. for C30H27NO6: C, 72.42; H, 5.47; N, 2.82. Found: C, 72.25; H, 5.55;
N,2.94.
Synthesis
of
methyl
3,5-dihydroxy-4-(2-oxo-2-(phenylamino)ethoxy)benzoate (9). Under ice bath, 8
(498 mg, 1 mmol), 10% Pd/C (110 mg), methanol (10 mL) were added to a flask and
placed under H2. The reaction mixture was stirred for 4 h at room temperature. After
filtration, the filtrate was concentrated and chromatographed on a silica gel column
(100-200 mesh) with a mixture of petroleum ether and acetone (4/1, v/v) as an eluent
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to give pure compound 9 as a white solid (210 mg, 66%). 9: mp 199-200 oC; 1H NMR
(d6-acetone/300 MHz) δ 9.60 (s, 1H), 9.23 (s, 2H), 7.68 (d, J = 7.5Hz, 2H), 7.35 (t, J
= 7.5Hz, 2H), 7.13 (t, J = 7.5Hz, 1H), 7.11 (s, 2H), 4.82 (s, 2H), 3.82 (s, 3H); 13C{1H}
NMR (d6-acetone/75 MHz) δ 170.5, 166.8, 151.5, 140.2, 139.0, 129.8, 127.6, 125.1,
120.6, 120.5, 110.1, 72.8, 52.3; IR (KBr) ν 3419 3330, 1706, 1601 cm-1; MS (EI-MS)
m/z 224 [M-PhNH2-H]+ (100%), 317 [M]+ (5); Anal. Calcd. for C16H15NO6: C, 60.57;
H, 4.77; N, 4.41. Found: C, 60.59; H, 4.95; N, 4.26.
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General procedure for synthesis of 2. Fine anhydrous potassium carbonate
powder (2 mmol) and acetonitrile (40 mL) were mixed in a flask and heated at reflux.
The suspension of 6 (0.4 mmol) and 3 (0.4 mmol) in acetonitrile (40 mL) was then
added. The resulting mixture was refluxed for another 15 min and cooled to room
temperature. After filtration, the filtrate was concentrated and chromatographed on a
silica gel column (100-200 mesh) and followed by recrystallization if necessary to
give pure compound 2 as a white solid.
2a. Chromatography with a mixture of petroleum ether, chloroform and acetone
(7/6/1, v/v/v) and recrystallized from hexane and acetone. 20% yield. mp 263-264 oC;
1H NMR (d6-acetone/300 MHz) δ 9.01 (s, 2H), 7.72 (dd, J = 3.2, 7.9 Hz, 2H),
7.63-7.61 (m, 8H), 7.39-7.25 (m, 8H), 7.13-7.07 (m, 4H), 4.62 (s, 4H), 3.85 (s, 6H);
13C{1H} NMR (d6-acetone/75 MHz) δ 175.1, 174.5, 166.2, 166.1, 164.9, 150.0,
147.6, 145.4, 138.9, 138.8, 134.4, 130.0, 129.6, 128.9, 127.0, 125.0, 124.7, 123.5,
120.9, 120.8, 116.4, 73.2, 52.8; IR (KBr) ν 3400, 1729, 1566, 1362, 1212 cm-1; MS
(MALDI-TOF) m/z (%): 1151.2 [M+Na]+ (45), 1153.2 [M+Na]+ (100), 1155.2
[M+Na]+ (55). Anal. Calcd. for C50H34Br2N8O14: C, 53.11; H, 3.03; N, 9.91. Found:
C, 53.26; H, 3.10; N, 9.83.
2b. Chromatography with a mixture of petroleum ether, chloroform and acetone
1
(8/6/1, v/v/v). 62% yield. mp >300oC; H NMR (CDCl3/300 MHz) δ 8.27 (s, 2H),
7.70 (s, 4H), 7.54-7.51 (m, 8H), 7.23 (t, J = 7.8 Hz, 4H), 7.08 (q, J = 8.1 Hz, 4H),
4.56 (s, 4H), 3.90 (s, 6H); 13C{1H} NMR (CDCl3/75 MHz) δ 173.5, 173.5, 164.7,
164.3, 146.5, 145.7, 144.1, 136.7, 132.9, 129.3, 129.0, 127.2, 125.1, 123.2, 120.3,
117.4, 72.8, 52.9; IR (KBr) ν 3405, 3279, 1732, 1564, 1360, 1224 cm-1; MS
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(MALDI-TOF) m/z (%): 1308.8 [M+Na]+ (60), 1310.8 [M+Na]+ (100), 1312.9
[M+Na]+ (70). Anal. Calcd. for C50H32Br4N8O14: C, 46.61; H, 2.50; N, 8.70. Found:
C, 46.52; H, 2.52; N, 8.55.
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2c. Chromatography with a mixture of petroleum ether, chloroform and acetone
(7/6/1, v/v/v). 17% yield. mp 184-185 oC; 1H NMR (d6-acetone /300 MHz) δ 9.03 (s,
2H), 7.93 (dd, J = 3.1, 7.9 Hz, 2H), 7.64-7.61 (m, 8H), 7.39-7.25 (m, 6H), 7.14-7.03
(m, 6H), 4.63 (s, 4H), 3.85 (s, 6H); 13C{1H} NMR (d6-acetone/75 MHz) δ 175.1,
174.5, 166.2, 166.1, 164.9, 153.1, 147.6, 145.5, 140.6, 138.9, 138.8, 130.8, 129.6,
129.0, 127.1, 125.0, 123.9, 123.5, 121.0, 120.9, 90.4, 73.2, 52.8; IR (KBr) ν 3479,
1727, 1566, 1365, 1203 cm-1; MS (MALDI-TOF) m/z (%): 1247.1 [M+Na]+ (100),
1249.1 [M+Na]+ (12). Anal. Calcd. for C50H34I2N8O14: C, 49.04; H, 2.80; N, 9.15.
Found: C, 48.93; H, 2.91; N, 8.93.
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2d. Chromatography with a mixture of petroleum ether, chloroform and acetone
(8/6/1, v/v/v) and recrystallized from cyclohexane and dichloromethane. 40% yield.
mp 294-295 oC; 1H NMR (d6-acetone/300 MHz) δ 8.95 (s, 2H), 7.93 (br, 4H), 7.67 (s,
4H), 7.62 (d, J = 7.8 Hz, 4H), 7.24 (t, J = 7.8 Hz, 4H), 7.08 (t, J = 7.3 Hz, 2H), 6.90
(t, J = 7.9 Hz, 2H), 4.64 (s, 4H), 3.85 (s, 6H); 13C{1H} NMR (d6-acetone/125 MHz) δ
174.5, 174.1, 165.7, 164.9, 152.8, 147.1, 145.4, 140.9, 138.8, 130.8, 129.7, 127.2,
125.0, 123.5, 121.0, 90.9, 73.1, 52.8; IR (KBr) ν 3300, 1730, 1571, 1356, 1215 cm-1;
MS (MALDI-TOF) m/z (%): 1498.9 [M+Na]+ (100), 1499.9 [M+Na]+ (47). Anal.
Calcd. for C50H32I4N8O14: C, 40.67; H, 2.18; N, 7.59. Found: C, 40.71; H, 2.31; N,
7.49.
Synthesis of 11. Fine anhydrous potassium carbonate powder (154 mg, 1.1
mmol), phenol (105 mg, 1.1 mmol) and acetonitrile (25 mL) were mixed in a flask
and heated at reflux for 30 mins. The suspension of 10 (0.386 g, 0.5 mmol) in
acetonitrile (45 mL) was then added dropwise within 35 mins. The resulting mixture
was refluxed for another 35 mins and cooled to room temperature. After filtration, the
filtrate was concentrated and chromatographed on a silica gel column (100-200 mesh)
with a mixture of petroleum ether and ethyl acetate (3/1, v/v) as an eluent to give pure
o
1
compound 11 as a white solid (228 mg, 51%). 11: mp 113-114 C; H NMR
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(CDCl3/500 MHz) δ 7.56 (s, 4H), 7.44 (t, J = 7.9 Hz, 4H), 7.31 (t, J = 7.4 Hz, 2H),
7.23 (t, J = 7.4 Hz, 2H), 7.16 (t, J = 7.6 Hz, 4H), 7.12 (d, J = 7.8 Hz, 4H), 7.07 (d, J =
7
8
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7.5 Hz, 4H), 4.91 (s, 4H), 3.85 (s, 6H); C{1H} NMR (CDCl3/125 MHz) δ 174.3,
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173.2, 165.0, 151.7, 146.8, 145.0, 135.9, 129.8, 128.4, 128.3, 127.6, 126.5, 125.8,
122.2, 121.5, 75.6, 52.6; IR (KBr) ν 1727, 1598, 1362, 1200 cm-1; HRMS (ESI): m/z
calcd. for [M+H]+ C48H35N6O12: 887.2307; found 887.2287.
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Synthesis of 12. At room temperature, 11 (355 mg, 0.4 mmol), anhydrous
aluminum chloride (1.092 g, 8 mmol) and dried toluene (40 mL) were mixed in a
flask. The mixture was stirred for 3.5 h under argon gas and poured into ice water (50
mL), then extracted with ethyl acetate (2×40 mL). The organic phase was dried with
anhydrous sodium sulfate and filtered. After removal of organic solvent, the residue
was recrystallized with dichloromethane and hexane to give pure compound 12 as a
o
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white solid (215 mg, 76%). 12: mp 224-225 C; H NMR (d6-acetone/500 MHz) δ
9.66 (s, 1H), 7.52 (t, J =1.5, 7.8 Hz, 4H), 7.47 (s, 4H), 7.35 (t, J =7.5 Hz, 2H), 7.32 (d,
13
J =7.7 Hz, 4H), 3.78 (s, 6H); C{1H} NMR (d6-acetone/125 MHz) δ 175.4, 174.4,
165.4, 152.9, 147.2, 141.2, 130.6, 127.1, 122.7, 122.5, 121.7, 52.4; IR (KBr) ν 3324,
1721, 1568, 1368, 1199 cm-1; HRMS (ESI): m/z calcd. for [M-H]- C34H21N6O12:
705.1223; found 705.1218.
ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding Author
qiqiangw@iccas.ac.cn; dxwang@iccas.ac.cn
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
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This work was financially supported by NNSFC (21772203, 21761132024,
21502200, 21521002), Chinese Academy of Sciences (QYZDJ-SSW-SLH023) and
the Project for Extramural Scientists of the State Key Laboratory of
Agrobiotechnology (grant 2018SKLAB6-8). Q.-Q.W. also thanks the Thousand
Young Talents Program for support.
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Supporting Information. Experimental details for binding studies, evaluation of ion
1
transport activity and anticancer activity. Copies of H and 13C NMR spectra, and
crystallographic data. CCDC 1892121 and 1892122 contain the supplementary
crystallographic data for this paper. This material is available free of charge via the
Internet at http://pubs.acs.org.
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