Synthesis and biological activity of squaramido-tethered bisbenzimidazoles as synthetic anion transporters

Article abstract of DOI:10.1039/d0ra10189c

A series of squaramido-tethered bisbenzimidazoles were synthesized from the reaction of diethyl squarate with substituted 2-aminomethylbenzimidazoles. These conjugates exhibit moderate binding affinity toward chloride anions. They are able to facilitate the transmembrane transport of chloride anions most probably via an anion exchange process, and tend to be more active at acidic pH than at physiological pH. The viability of these conjugates toward four selected solid tumor cell lines was evaluated using an MTT assay and the results suggest that some of these conjugates exhibit moderate cytotoxicity probably in an apoptotic fashion.

Full text of DOI:10.1039/d0ra10189c

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Synthesis and biological activity of squaramido-
tethered bisbenzimidazoles as synthetic anion
transporters†
Cite this: RSC Adv., 2021, 11, 3972
a
b
a
a
Zhong-Kun Wang, Xiao-Qiao Hong, Jinhui Hu, Yuan-Yuan Xing
ac
and Wen-Hua Chen
*
A series of squaramido-tethered bisbenzimidazoles were synthesized from the reaction of diethyl squarate
with substituted 2-aminomethylbenzimidazoles. These conjugates exhibit moderate binding affinity toward
chloride anions. They are able to facilitate the transmembrane transport of chloride anions most probably
via an anion exchange process, and tend to be more active at acidic pH than at physiological pH. The
viability of these conjugates toward four selected solid tumor cell lines was evaluated using an MTT
assay and the results suggest that some of these conjugates exhibit moderate cytotoxicity probably in an
apoptotic fashion.
Received 3rd December 2020
Accepted 13th January 2021
DOI: 10.1039/d0ra10189c
rsc.li/rsc-advances
via hydrogen-bonding interactions. Some simple squar-
amides, such as triuoromethylphenyl derivatives have been
1
. Introduction
During the past decades, substantial interest has been shown in found to efficiently facilitate the transport of chloride anions
3
2
the creation of small-molecule, organic compounds that are across phospholipid bilayers. More interestingly, it has been
able to facilitate the transport of anions, in particular chloride demonstrated that these squaramide derivatives are able to
1
–10
anions across cellular phospholipid membranes.
called synthetic anion transporters may serve as useful somes, inhibiting Cathepsin B enzyme activity and disturbing
These so- trigger the apoptosis of cancer cell lines by deacidifying lyso-
2
4
research tools to clarify the structures and functions of natural autophagy. Replacement of the oxygen atoms in squaramides
anion channels and transporters. More interestingly, because with sulfur atoms (to give thiosquaramides) leads to pH
anion transporters are able to perturb the chemical gradients dependent anion transport.
2
6,27
Such pH-regulating anion
within cells to trigger apoptosis, they may be developed as transporters may serve as attractive candidates for the devel-
a novel class of chemotherapeutic agents for the treatment of opment of anticancer drugs. Attachment of squaramido
11–20
cancers.
peutic potentials for channelopathies such as cystic brosis anion receptors that show promising anion transport. As for
In addition, they may also have high chemothera- substituents to a rigid choloyl unit leads to extremely strong
28
(
CF) by restoring the ux of anions through epithelial cell (benz)imidazole-based anion transporters, it has been re-
20,21
membranes in CF patients.
As such, more small-molecule ported that incorporation of (benz)imidazolyl or (benz)imi-
anion transporters with diverse structures and promising bio- dazolium groups into the structures of anion transporters may
1
–10
logical activity are in demand.
In these aspects, squaramides and (benz)imidazoles activity.
represent two classes of ideal structural skeletons for the strong antibacterial properties of some benzimidazolium-
be favorable to the anion transport efficiency and biological
3
3–40
For example, Schmitzer et al. have described the
2
2–40
construction of superior anion transporters.
ascribed to their unique features, including ready availability, their ability to insert into the cellular membranes and alter the
high stability and strong capability to complex anions mainly membrane permeability for chloride anions. We have also
shown in our previous studies that 1,3-bis(benzimidazol-2-yl)
This may be based anion transporters, which is a likely consequence of
3
8
benzene and its derivatives exhibit potent anion transport
a
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, P. R.
with promising anti-proliferative activity towards the selected
China. E-mail: whchen@wyu.edu.cn
b
4
1–43
solid tumor cell lines.
School of Pharmaceutical Sciences, Tsinghua University, Haidian District, Beijing
1
00084, P. R. China
These studies have raised the feasibility that conjugation of
c
State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical squaramides with benzimidazoles may lead to a novel class of
Genomics, Peking University Shenzhen Graduate School, Shenzhen 518055, P. R.
strong anion receptors. To test this hypothesis, we synthesized
a squaramide-tethered bisbenzimidazole 1 (Fig. 1). In this
China
†
Electronic
supplementary
information
(ESI)
available:
Structural
conjugate, the two benzimidazolyl subunits are linked to the
squaramido moiety via a CH unit so that the target molecule
characterization and experimental data for the anion recognition, anion
transport and biological activity of each compound. See DOI: 10.1039/d0ra10189c
2
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Fig. 1 Structures of compounds 1–14 and the interactions of compounds 1–13 with chloride anions.
is exible enough to complex anions through induced-t,
2
. Results and discussion
cooperative interactions with both the benzimidazolyl and
4
4–46
2.1 Synthesis
squaramido NHs (Fig. 1).
In addition, inspired by our
previous ndings that introduction of electron-withdrawing Compounds 1–14 were synthesized as shown in Scheme 1.
substituents onto the benzimidazolyl subunits of 1,3- Reaction of diethyl squarate with substituted 2-amino-
bis(benzimidazol-2-yl)benzene is favorable to the anion methylbenzimidazoles 28–40 gave compounds 1–13. Reaction
4
2,43
transport,
compound 1 with diverse substituents (to give compounds 2– 41) and subsequent reaction with n-propylamine afforded
3) with the aim at optimizing the anionophoric activity. For compound 14. Compounds 28–40 were prepared starting from
comparison, we also prepared compound 14 bearing only one the condensation of N-Boc-gly with substituted 1,2-dia-
we modied the benzimidazolyl subunits of of compound 28 with excess diethyl squarate (to give compound
1
47,48
benzimidazolyl subunit. Herein we report the synthesis of minobenzene using the procedures previously reported.
compounds 1–14 as well as their anion transport properties Compounds 1–14 were characterized by means of NMR ( H and
and anti-proliferative activity toward some solid tumor cell
lines.
1
1
3
C) and ESI MS (LR and HR) (see Experimental section and
Fig. S1–S56†).
Scheme 1 Synthesis of compounds 1–14.
ꢁ
3
ꢁ2
1
Fig. 2 (a) Negative ESI MS spectrum of compound 1 (1.0 ꢀ 10 M) mixed with TBACl (1.5 ꢀ 10 M) in 4 : 1 CH
3
CN-DMSO. (b) H NMR spectra of
ꢁ3
compound 1 (1.0 ꢀ 10 M) in the presence of TBACl of varying concentrations, in 4 : 1 CD
3 6
CN-DMSO-d .
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2
.2 Anion recognition
To explore the anion binding properties of compounds 1–14, we
rstly measured the electrospray ionization (ESI) mass spectra compounds 1–14 are able to complex chloride anions, they may
2.3 Anion transport
2.3.1 Chloride transport at physiological pH. As

of a mixture of compounds 1–14 with tetra(n-butyl)ammonium be capable of facilitating the transport of chloride anions across
chloride (TBACl). As shown in Fig. 2a and S57 and S58,† in phospholipid membranes. To address this, we measured the
addition to the ion peaks from compounds 1–14 (for example, efflux of chloride anions out of egg-yolk L-a-phosphatidylcho-
ꢁ
m/z ¼ 371.17, [1 ꢁ H] , Fig. 2a), new ion peaks that are line (EYPC) vesicles (100 nm, extrusion) using chloride ion
49
assignable to the 1 : 1 complexes with chloride anions were selective electrode techniques (Fig. 3a and S74–S87†). The
ꢁ
observed (for example, m/z ¼ 406.86, [1 + Cl] , Fig. 2a). These results suggest that the efflux of chloride anions across the
observations suggest that compounds 1–14 are able to form liposomal membranes is facilitated in the presence of conju-
stable 1 : 1 complexes with chloride anions.
gates 1–13. Expectedly, compound 14 is not active in facilitating
To clarify the binding sites and affinity of compounds 1–14 the transport of chloride anions (Fig. S87†). To quantitatively
1
toward chloride anions, we carried out H NMR titrations with characterize the transport efficiency of compounds 1–13 toward
TBACl (Fig. 2b and S59–S71†). Upon the addition of TBACl, both chloride anions, we carried out concentration-dependent chlo-
the benzimidazolyl and squaramido NHs were gradually ride efflux experiments and used a Hill equation to analyze the
downeld shied simultaneously, suggesting that they are relationship between the relative chloride efflux at 260 s and the
involved in the recognition of chloride anions, probably via concentration of each compound. This analysis gives the EC50
hydrogen bonding interactions (Fig. 1). To quantitatively char- value, the concentration that each compound needs to reach
50
acterize the affinity, we measured the association constants 50% of the maximum efflux.
's) of compounds 1–14 with chloride anions, by nonlinear As shown in Table 1, compounds 1–13 exhibit chloride
(K
a
curve tting the relationship between the downeld shis of the transport efficiency with the EC50 values ranging from
squaramido NHs and the concentrations of TBACl based on 1.84 mol% to 58.0 mol% at pH 7.0, suggesting that they are
a 1 : 1 model (Fig. S72 and S73†). The results are listed in Table modest anion transporters. Calcein leakage experiments based
1
and suggest that compounds 1–13 exhibit moderate affinity on compounds 1–3 and 5–6 indicate that the transport of
toward chloride anions. Compound 14 is weaker than chloride anions across the membranes was triggered by the
51
compounds 1–13, which suggests that a second benzimidazolyl presence of these compounds (Fig. S88†). Careful analysis of
subunit is necessary for achieving stronger anion recognition. the EC50 values afforded several observations on the structure–
Structural optimization of compound 1 with diverse substitu- activity relationships. Firstly, the observation that compound 14
ents led to up to 10-fold increase in the binding affinity toward exhibits negligible anion transport activity, suggests that two
chloride anions, suggesting that the anion-binding affinity may benzimidazolyl subunits are required in the chloride transport.
be regulated by the substituents.
Secondly, the substituents exhibit obvious effects on the
transport efficiency of compounds 1–13. Compared with
Table 1 Lipophilicity (clog P), anion binding affinity and chloride efflux efficiency of compounds 1–14
b
c
Anion binding
Chloride efflux efficiency (EC50, mol%)
a
ꢁ1
d
e
f
Compound
clog P
K
a
(M
)
RA
1
pH 7.0
pH 6.0
pH 5.0
pH 4.0
RA
2
RA
3
2
2
2
2
2
2
2
2
2
2
3
2
2
1
2
3
4
5
6
7
8
9
1.52
2.55
1.21
5.03
1.40
2.09
2.73
3.06
2.09
1.81
3.28
2.09
1.81
1.32
(1.31 ꢂ 0.25) ꢀ 10
(2.92 ꢂ 0.32) ꢀ 10
(3.67 ꢂ 0.67) ꢀ 10
(4.34 ꢂ 0.49) ꢀ 10
(1.52 ꢂ 0.91) ꢀ 10
(3.70 ꢂ 0.22) ꢀ 10
(2.97 ꢂ 0.83) ꢀ 10
(2.54 ꢂ 0.18) ꢀ 10
(2.41 ꢂ 0.30) ꢀ 10
(2.84 ꢂ 0.26) ꢀ 10
(1.26 ꢂ 0.34) ꢀ 10
(3.71 ꢂ 0.33) ꢀ 10
(2.37 ꢂ 0.32) ꢀ 10
67.3 ꢂ 20.7
1.9
4.3
5.5
6.4
2.3
5.5
4.4
3.8
3.6
4.2
18.7
5.5
3.5
1.0
16.7 ꢂ 2.83
3.78 ꢂ 0.72
6.67 ꢂ 0.79
50.4 ꢂ 22.9
1.84 ꢂ 0.35
6.41 ꢂ 0.62
31.5 ꢂ 10.4
47.8 ꢂ 15.7
58.0 ꢂ 20.3
23.3 ꢂ 2.88
48.9 ꢂ 16.4
28.0 ꢂ 7.80
13.9 ꢂ 1.80
NA
10.9 ꢂ 1.10
3.72 ꢂ 0.65
6.08 ꢂ 0.77
37.3 ꢂ 11.1
1.76 ꢂ 0.16
5.60 ꢂ 0.47
21.4 ꢂ 3.58
23.5 ꢂ 2.89
20.7 ꢂ 2.72
20.5 ꢂ 4.52
41.1 ꢂ 14.0
14.4 ꢂ 2.30
10.2 ꢂ 1.16
NA
9.12 ꢂ 1.13
1.93 ꢂ 0.21
3.74 ꢂ 0.50
26.7 ꢂ 9.52
1.38 ꢂ 0.12
2.80 ꢂ 0.24
15.4 ꢂ 3.73
16.7 ꢂ 4.04
11.0 ꢂ 1.94
10.15 ꢂ 1.48
30.5 ꢂ 12.6
10.00 ꢂ 1.47
6.10 ꢂ 0.84
NA
1.64 ꢂ 0.50
0.27 ꢂ 0.09
1.15 ꢂ 0.48
21.4 ꢂ 10.6
0.44 ꢂ 0.07
1.99 ꢂ 0.63
6.84 ꢂ 2.98
2.77 ꢂ 1.03
1.82 ꢂ 0.57
5.99 ꢂ 2.31
27.2 ꢂ 23.4
6.06 ꢂ 0.80
3.38 ꢂ 0.79
NA
1.0
4.4
2.5
0.3
9.1
2.6
0.5
0.3
0.3
0.7
0.3
0.6
1.2
NA
10.2
14.0
5.8
2.4
4.2
3.2
4.6
17.3
31.9
3.9
10
11
12
13
14
1.8
4.6
4.1
NA
a
b
1
Calculated using MarvinSketch (Version 6.1.0, Weighted Model, ChemAxon, MA). Estimated by means of H NMR titrations in CD
4/1, v/v). Measured by means of chloride ion selective electrode techniques, under the conditions of an intravesicular 500 mM NaCl solution
3
CN-DMSO-d
6
c
(
(
d
25 mM HEPES buffer) and extravesicular 500 mM NaNO
¼ K
compound at pH 7 relative to compound 1 at pH 7, that is, RA
3
solution (25 mM HEPES buffer). RA
(each compound)/K
1
denotes the anion binding affinity of each
e
compound relative to compound 14, that is, RA
1
a
a
(compound 14)
.
RA denotes the chloride efflux efficiency of each
2
f
2
¼ EC50 (compound 1)/EC50 (each compound)
.
3
RA denotes the chloride efflux
efficiency of each compound at pH 4 relative to itself at pH 7, that is, RA
3
¼ EC50 (pH 7)/EC50 (pH 4)
.
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Fig. 3 (a) Relative efflux of chloride anions out of EYPC liposomes enhanced by compounds 1–14 (5 mol%) under the conditions of an intra-
vesicular 500 mM NaCl solution in 25 mM HEPES buffer (pH 7.0) and extravesicular 500 mM NaNO in 25 mM HEPES buffer (pH 7.0). (b) Relative
efflux of chloride anions out of EYPC liposomes enhanced by compound 1 (5 mol%) at pHs varying from 4.0 to 7.0, under the conditions of an
intravesicular 500 mM NaCl solution in 25 mM HEPES buffer (pH 7.0) and extravesicular 500 mM NaNO in 25 mM HEPES buffer (pH 7.0).
3
3
compound 1, compounds 2, 3, 5, 6 and 13 exhibit 2.1–9.1-fold whether compounds 1–13 are able to facilitate the transport of
higher anion transport, whereas the other compounds are less chloride anions in a pH dependent fashion. To address this, we
active. This result suggests that the transport efficiency may be measured the efflux of chloride anions out of the EYPC lipo-
moderately regulated by the substituents. Thirdly, the highest somes at pHs varying from 4.0 to 7.0 (Fig. 3b and S74–S87,†
chloride transport was observed in compound 5 bearing nitro Table 1). The EC50 value for each compound increases by 2 to 32
groups on the benzimidazolyl subunits, ca. 9-fold more active folds when the pH increases from 4 to 7, suggesting that all the
than compound 1. The other conjugates bearing electron- conjugates tend to be more active at lower pHs. Interestingly,
withdrawing substituents, in particular triuoromethyl groups the pH-dependent increment in the activity is greater for the
(i.e., compounds 4 and 11) do not show higher activity than derivatives that are less active at physiological pH (e.g.,
compound 1. These results, together with the nding that the compounds 8 and 9) than for those that are more active at
conjugates bearing methoxy or methyl groups on the benzimi- physiological pH (e.g., compounds 2, 3, 6 and 13). It should be
dazolyl subunits exhibit higher chloride transport, suggest that noted that compound 14 exhibits negligible activity in the pH
introducing electron-withdrawing substituents on the benzi- range from 4 to 7.
midazolyl subunits may not necessarily lead to an increase in
2.3.3 Probable mechanism of action of anion transport. To
the activity. This is apparently different from our the ndings clarify the probable mechanism of action by which compounds
that electron-withdrawing groups on the benzimidazolyl 1–13 facilitate the transmembrane transport of chloride anions,
subunits of 1,3-bis(benzimidazol-2-yl)benzene are favourable to we measured the efflux of chloride anions in the presence of
4
1–43
53,54
the chloride transport.
.3.2 Effect of pH on the chloride transport. Literature Fig. 4a, b and S89–S92,† the efflux of chloride anions is regu-
reports show that benzimidazolyl derivatives exhibit pH lated by the anions rather than by the metal ions, which
different alkali metal ions or anions.
As can be seen from
2
52
dependent anion transport. This encouraged us to explore suggests that an anion exchange process may be involved in the
+
+
+
+
+
Fig. 4 (a) Relative chloride efflux out of EYPC liposomes containing Li , Na , K , Rb or Cs , enhanced by compound 1 (5 mol%), under the
conditions of an intravesicular 500 mM MCl solution in 25 mM HEPES buffer (pH 7.0) and extravesicular 500 mM MNO in 25 mM HEPES buffer
pH 7.0) (M ¼ Li, Na, K, Rb or Cs). (b) Relative chloride efflux out of EYPC liposomes, enhanced by compound 1 (5 mol%), under the conditions of
an intravesicular 500 mM NaCl in 25 mM HEPES buffer (pH 7.0) and extravesicular 500 mM NaNO , 500 mM NaHCO or 250 mM Na SO in
5 mM HEPES buffer (pH 7.0). (c) Chloride transport across a bulky nitrobenzene membrane, promoted by compound 1 (1.0 mM) and detected by
a chloride ion selective electrode in the receiving aqueous phase of U-tube, under the conditions of 500 mM NaNO (25 mM HEPES, pH 7.0) for
in the nitrobenzene organic
3
(
3
3
2
4
2
3
the U-tube chloride receiver, 500 mM NaCl (25 mM HEPES, pH 7.0) for the U-tube chloride donor and 2 mM TBAPF
phase.
6
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Table 2 Inhibitory activity of compounds 4, 5, 7, 8, 11 and doxorubicin a positive drug. The results indicate that some of these conju-
gates, such as compounds 4, 5, 7, 8 and 11 exhibit moderate
inhibitory activity toward the four cancer cells. It appears that
a
Inhibitory activity (IC50, mM)
Compound HeLa
A549
>50
MCF-7
HepG2
these conjugates exhibit no obvious cytotoxic effect on the LO2
cell line. In addition, we used ow cytometry to investigate the
probable mode of cell death triggered by compound 11 (Fig. 5).
The results imply that compound 11 may cause cell death via an
apoptotic fashion. More work will be carried out to clarify this.
4
5
7
8
1
23.27 ꢂ 2.19 >50
18.10 ꢂ 3.19 18.74 ꢂ 1.84
>50
12.62 ꢂ 2.30
>50
>50
37.31 ꢂ 6.07 9.85 ꢂ 1.72 16.09 ꢂ 4.52
31.94 ꢂ 13.36 >50
17.35 ꢂ 5.45 14.41 ꢂ 5.48
10.51 ꢂ 2.57 12.81 ꢂ 2.17
1
9.59 ꢂ 0.56
>50
Doxorubicin 0.99 ꢂ 0.16
a
1.99 ꢂ 0.08 0.77 ꢂ 0.10
1.75 ꢂ 0.12
3
. Conclusion
Calculated using GraphPad Prism v8.0.
In conclusion, we have successfully synthesized a series of
squaramido-tethered bisbenzimidazoles and assessed their
anion binding affinity, transport properties and biological
activity. These conjugates are able to complex chloride anions,
and facilitate the transport of chloride anions with moderate
pH dependence and via an anion exchange process. In addition,
some of these conjugates exhibit moderate anti-proliferative
effects on the selected solid tumor cell lines, whereas without
signicant cytotoxicity toward the normal cell line. Further work
will be carried out to optimize the structures of these conjugates
so that promising pH-regulated anionophoric activity may be
achieved. The outcomes will be reported in due course.
efflux of chloride anions. In addition, U-tube experiments show
that these conjugates are able to facilitate the transport of
chloride anions across bulky organic layers (Fig. 4c and S93†),
suggesting that compounds 1–13 function primarily as mobile
55
carriers.
2.4 Cell viability
The ability of compounds 1–13 to facilitate the transport of
anions across liposomal membranes encouraged us to investi-
gate the biological effect of each compound on four selected
solid cancer cell lines (that is, HeLa cervical cancer cells, A549
lung adenocarcinoma cells, MCF-7 breast cancer cells and
HepG2 human liver cancer cells) and one normal LO2 cell line.
4. Experimental
4.1 Generals
1
13
Using a conventional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- The H and C NMR spectra were measured on a Bruker Avance
diphenyltetra zolium bromide) assay, we measured the inhibi- AV 400 or 500 spectrometer, and the data were reported relative
tory activity of each compound at 50 mM toward those cell lines to the deuterium solvents. Waters UPLC/Quattro Premier XE
(Fig. S94†), and the IC50 values of those conjugates that exhibit and Bruker maXis 4G ESI-Q-TOF mass spectrometers were used
higher than 50% inhibition at the above-mentioned concen- to measure the LR and HR ESI-MS spectra, respectively.
trations (Fig. S94–S96† and Table 2). Doxorubicin was used as Analytical thin-layer chromatography (TLC) plates (silica gel,
Fig. 5 Flow cytometry of (a) untreated HeLa cells and (b–e) HeLa cells treated with 11 ((b) 10 mM; (c) 20 mM; (d) 30 mM) and doxorubicin ((e) 0.03
mM) for 24 h and stained with Annexin V-FITC and PI solution. (f) The apoptosis rate of HeLa cells by compound 11 at different concentrations
(
mean ꢂ s.d., n ¼ 3, *P < 0.05, One-Way ANOVA).
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ꢁ
GF254) were detected by use of iodine and UV (254 or 365 nm). negative HR-ESI-MS for C24
EYPC vesicles were prepared by extrusion through nuclepore 291.10878; found: 291.10983.
H
12
F
12
N
6
O
2
([M ꢁ H] ) calcd:
track-etched polycarbonate membranes (100 nm, Whatman,
Florham Park, New Jersey, USA) on an Avanti's Mini-Extruder compound 15. 342 mg (34%). H NMR (400 MHz, CD OD) d 7.05
4.2.6 Compound 20. Similar procedures as described for
1
3
(
Avanti Polar Lipids, Inc., Alabaster, Alabama, USA). Chloride (d, J ¼ 5.4 Hz, 1H), 6.84–6.80 (m, 1H), 4.47 (s, 2H), 1.47 (s, 9H)
ꢁ
efflux was measured by using a Mettler-Toledo PerfectIon™ and negative ESI-MS: m/z 281.88 ([M ꢁ H] ).
chloride ion selective electrode assembled with a Mettler-
Toledo Seven Compact S220 ionometer.
4.2.7 Compound 21. Similar procedures as described for
1
3
compound 15. 140 mg (14%). H NMR (CD OD, 400 MHz)
EYPC and MTT were purchased from Sigma Chemical Co. (St d 7.51–7.46 (m, 2H), 7.19 (d, J ¼ 8.5 Hz, 1H), 4.47 (s, 2H), 1.46 (s,
ꢁ
Louis, USA). All the other chemicals and reagents were obtained 9H) and negative ESI-MS: m/z 280.12 ([M ꢁ H] ).
from commercial sources and used without further purication.
The experimental protocols for the measurement of anion compound 15. 221 mg (38%). H NMR (400 MHz, CD OD) d 7.67
4.2.8 Compound 22. Similar procedures as described for
1
3
recognition, anion transport and biological activity were (s, 1H), 7.42 (s, 1H), 7.32 (d, J ¼ 8.4 Hz, 1H), 4.42 (s, 2H), 1.46 (s,
1
3
included in the ESI.†
9H); C NMR (100 MHz, CD
8
3
OD) d 158.5, 155.9, 129.1, 123.8,
ꢁ
1.0, 39.6, 28.9; negative ESI-MS: m/z 323.81 ([M ꢁ H] ) and
ꢁ
negative HR-ESI-MS for C13
24.03421; found: 324.03513.
H16BrN
3
O
2
([M ꢁ H] ) calcd:
4
.2 Synthesis of compounds 15–27
.2.1 Compound 15. A solution of N-Boc-gly (805 mg, 4.59
mmol), EDC (1.77 g, 9.23 mmol), HOBt (936 mg, 6.92 mmol) and compound 15. 365 mg (37%). H NMR (400 MHz, CD
DMAP (30 mg, 0.24 mmol) in CH Cl
(40 mL) was stirred at (d, J ¼ 8.3 Hz, 1H), 6.81 (t, J ¼ 10.3 Hz, 1H), 4.46 (s, 2H), 1.45 (s,
room temperature for 10 min, and added drop wise to a solution 9H); C NMR (100 MHz, CD
3
4
4.2.9 Compound 23. Similar procedures as described for
1
OD) d 7.05
3
2
2
1
3
3
OD) d 161.5 (d, J ¼ 42.5 Hz), 159.1
of 1,2-diaminobenzene (500 mg, 4.62 mmol) in DMF (1.5 mL). (d, J ¼ 63.4 Hz), 158.1, 156.0, 129.7, 98.7, 98.5, 98.4, 98.2, 80.8,
ꢁ
The resulting mixture was stirred at room temperature for 3 39.2, 28.7; negative ESI-MS: m/z 281.91 ([M ꢁ H] ) and negative
ꢁ
d and concentrated under reduced pressure. The obtained HR-ESI-MS for C13
residue was dissolved in EtOAc (20 mL) and washed subse- found: 282.10547.
H
15
F
2
N
3
O
2
([M ꢁ H] ) calcd: 282.10485;
quently with brine (20 mL ꢀ 2), saturated aqueous NH
solution (20 mL ꢀ 2), saturated aqueous NaHCO
solution compound 15. 143 mg (23%). H NMR (400 MHz, CD
20 mL ꢀ 2) and brine (20 mL). The organic layer was dried over (d, J ¼ 7.3 Hz, 1H), 7.19–7.14 (m, 1H), 6.92 (t, J ¼ 9.2 Hz, 1H),
4
Cl
4.2.10 Compound 24. Similar procedures as described for
1
OD) d 7.30
3
3
(
1
3
2 4
anhydrous Na SO
3
, and concentrated under reduced pressure. 4.39 (s, 2H), 1.37 (s, 9H); C NMR (100 MHz, CD OD) d 158.4,
The obtained residue was dissolved in acetic acid (5 mL). The 155.0, 124.1 (d, J ¼ 27.2 Hz), 108.4 (d, J ¼ 69.1 Hz), 80.9, 39.6,
ꢃ
ꢁ
resulting solution was heated at 70 C for 10 h, and concen- 28.5; negative ESI-MS: m/z 263.93 ([M ꢁ H] ) and negative HR-
ꢁ
trated under reduced pressure. The obtained residue was dis- ESI-MS for C13
solved in ether (20 mL) and ltered. The ltrate was 264.11514.
H
16FN
3
O
2
([M ꢁ H] ) calcd: 264.11428; found:
concentrated under reduced pressure and the obtained residue
4.2.11 Compound 25. Similar procedures as described for
1
was puried by chromatography on a silica gel column (petro- compound 15. 246 mg (28%). H NMR (400 MHz, CD
OD) d 7.83
3
leum ether/EtOAc, 2/1, v/v) to give compound 15 (515 mg, 45%) (s, 1H), 7.66 (d, J ¼ 7.9 Hz, 1H), 7.49 (d, J ¼ 8.3 Hz, 1H), 4.52 (s,
1
ꢁ
having H NMR (400 MHz, CD
3
OD) d 7.51 (s, 2H), 7.20 (s, 2H), 2H), 1.47 (s, 9H) and negative ESI-MS: m/z 314.08 ([M ꢁ H] ).
4
.48 (s, 2H), 1.47 (s, 9H) and negative ESI-MS: m/z 245.96 ([M ꢁ
4.2.12 Compound 26. Similar procedures as described for
ꢁ
1
H] ).
compound 15. 159 mg (16%). H NMR (400 MHz, CD
3
OD) d 7.29
OD)
1
3
4
.2.2 Compound 16. Similar procedures as described for (s, 2H), 4.36 (s, 2H), 1.37 (s, 9H); C NMR (100 MHz, CD
compound 15. 266 mg (25%). H NMR (400 MHz, CD
3
1
3
OD) d 7.38 d 158.4, 156.4, 150.3 (d, J ¼ 61.6 Hz), 147.9 (d, J ¼ 62.4 Hz),
(
d, J ¼ 7.2 Hz, 1H), 7.30 (s, 1H), 7.03 (d, J ¼ 7.6 Hz, 1H), 4.45 (s, 132.4, 129.9, 106.2, 100.5, 80.9, 39.6, 28.7; negative ESI-MS: m/z
ꢁ
2
(
H), 2.42 (s, 3H), 1.46 (s, 9H) and negative ESI-MS: m/z 259.83 281.94 ([M ꢁ H] ) and negative HR-ESI-MS for C13
H F N O
15 2 3 2
ꢁ
ꢁ
[M ꢁ H] ).
([M ꢁ H] ) calcd: 282.10485; found: 282.10583.
4
.2.3 Compound 17. Similar procedures as described for
4.2.13 Compound 27. Similar procedures as described for
1
1
3
3
compound 15. 458 mg (47%). H NMR (400 MHz, CD OD) d 7.38 compound 15. 268 mg (29%). H NMR (400 MHz, CD OD) d 7.84
(
2
(
d, J ¼ 8.8 Hz, 1H), 7.02 (s, 1H), 6.83 (d, J ¼ 8.8 Hz, 1H), 4.44 (s, (s, 1H), 7.67 (d, J ¼ 6.6 Hz, 1H), 7.50 (d, J ¼ 6.7 Hz, 1H), 4.53 (s,
ꢁ
H), 3.81 (s, 3H), 1.46 (s, 9H) and negative ESI-MS: m/z 275.83 2H), 1.48 (s, 9H) and negative ESI-MS: m/z 263.93 ([M ꢁ H] ).
ꢁ
[M ꢁ H] ).
4
.2.4 Compound 18. Similar procedures as described for
1
4.3 Synthesis of compounds 28–40
compound 15. 302 mg (38%). H NMR (400 MHz, CD
3
OD) d 8.09
(
s, 1H), 7.76 (s, 1H), 4.57 (s, 2H), 1.47 (s, 9H) and negative ESI-
4.3.1 Compound 28. To a solution of compound 15 (60 mg)
in CH OH (3 mL) was added HCl aqueous solution (2 M, 3 mL).
3
ꢁ
MS: m/z 381.87 ([M ꢁ H] ).
4
.2.5 Compound 19. Similar procedures as described for The resulting solution was stirred at room temperature for 1 h
1
compound 15. 287 mg (30%). H NMR (400 MHz, CD OD) d 8.46 and concentrated under reduced pressure. The obtained
3
(
2
s, 1H), 8.17 (d, J ¼ 8.9 Hz, 1H), 7.65 (d, J ¼ 7.1 Hz, 1H), 4.53 (s, residue was partitioned in ammonia solution (3 mL). The
ꢁ
H), 1.47 (s, 9H); negative ESI-MS: m/z 290.88 ([M ꢁ H] ) and mixture was stirred at room temperature for 15 min and
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concentrated under reduced pressure. This procedure was Then, a solution of diethyl squarate (55 mg, 0.32 mmol) in EtOH
repeated. Purication by preparative thin-layer chromatography (270 mL) was added. The resulting solution was stirred at room
(
CH Cl /MeOH/NH $H O, 24/3/1, v/v/v) afforded compound 28 temperature for 23 h. Purication by preparative thin-layer
2 2 3 2
1
(
2
39 mg, 45%). H NMR (500 MHz, CD OD) d 7.54 (dd, J ¼ 4.8, chromatography (CH Cl /CH OH, 20/1, v/v) afforded
3
2
2
3
1
.5 Hz, 2H), 7.22 (dd, J ¼ 4.8, 2.5 Hz, 2H), 4.03 (s, 2H).
3
compound 41 (44 mg, 71%) having H NMR (500 MHz, CD OD)
4
.3.2 Compound 29. Similar procedures as described for d 7.54 (dd, J ¼ 6.0, 3.2 Hz, 2H), 7.23 (dd, J ¼ 6.0, 3.2 Hz, 2H), 5.07
1
compound 28. 40 mg (72%) from compound 16 (90 mg). H (s, 2H), 3.59 (q, J ¼ 7.1 Hz, 2H), 1.16 (t, J ¼ 7.1 Hz, 3H); negative
ꢁ
NMR (400 MHz, CD
7
3
OD) d 7.40 (d, J ¼ 8.2 Hz, 1H), 7.31 (s, 1H), ESI-MS: m/z 270.09 ([M ꢁ H] ) and negative HR-ESI-MS for
ꢁ
.05 (d, J ¼ 8.2 Hz, 1H), 4.01 (s, 2H), 2.44 (s, 3H).
C
14
H
13
N
3
O
3
([M ꢁ H] ) calcd: 270.08542; found: 270.08732.
4
.3.3 Compound 30. Similar procedures as described for
1
compound 28. 55 mg (71%) from compound 17 (122 mg). H
4.5 Synthesis of compounds 1–13
NMR (400 MHz, CD OD) d 7.40 (d, J ¼ 8.8 Hz, 1H), 7.03 (d, J ¼
3
2
3
.3 Hz, 1H), 6.85 (dd, J ¼ 8.8, 2.4 Hz, 1H), 3.99 (s, 2H), 3.82 (s,
4.5.1 Compound 1. A solution of compound 28 (39 mg, 0.16
ꢃ
H).
mmol) and Et N (300 mL) in EtOH (3 mL) was stirred at 50 C for
3
4
.3.4 Compound 31. Similar procedures as described for 30 min. Then, a solution of diethyl squarate (7 mg, 0.042 mmol)
1
compound 28. 69 mg (70%) from compound 18 (134 mg). H in EtOH (100 mL) was added. The resulting solution was stirred
NMR (400 MHz, CD
ꢃ
3
OD) d 8.06 (s, 1H), 7.72 (s, 1H), 4.11 (s, 2H). at 50 C for 54 h. The formed precipitates were collected
4
.3.5 Compound 32. Similar procedures as described for through ltration under reduced pressure and washed with
1
compound 28. 62 mg (73%) from compound 19 (129 mg). H EtOH (20 mL ꢀ 5) to afford compound 1 (12 mg, 31%) having
1
NMR (400 MHz, CD OD) d 8.45 (d, J ¼ 2.0 Hz, 1H), 8.16 (dd, J ¼
6
H NMR (400 MHz, DMSO-d ) d 12.45 (s, 2H), 8.24 (s, 2H), 7.53
3
1
3
8
.9, 2.0 Hz, 1H), 7.65 (d, J ¼ 8.9 Hz, 1H), 4.09 (s, 2H).
(s, 4H), 7.17 (s, 4H), 5.02 (s, 4H); selected C NMR (125 MHz,
4
.3.6 Compound 33. Similar procedures as described for DMSO-d ) d 183.0, 167.7, 151.5, 121.7, 41.4; negative ESI-MS: m/z
6
1
ꢁ
compound 28. 42 mg (75%) from compound 20 (86 mg). H 371.07 ([M ꢁ H] ) and negative HR-ESI-MS for C H N O ([M
2
0
16 6 2
ꢁ
NMR (400 MHz, CD
.04 (s, 2H).
3
OD) d 7.28–7.25 (m, 1H), 7.16–7.09 (m, 1H), ꢁ H] ) calcd: 371.12510; found: 371.12570.
4
4.5.2 Compound 2. Similar procedures as described for
1
4.3.7 Compound 34. Similar procedures as described for compound 1; 35 mg (79%) from compound 29 (40 mg). H NMR
1
compound 28. 72 mg (85%) from compound 21 (132 mg). H (400 MHz, DMSO-d ) d 12.32 (s, 2H), 8.18 (s, 2H), 7.44–7.26 (m,
6
1
3
NMR (400 MHz, CD OD) d 7.68 (s, 1H), 7.44 (d, J ¼ 8.6 Hz, 1H), 4H), 6.99 (s, 2H), 4.99 (s, 4H), 2.39 (s, 6H); C NMR (100 MHz,
3
7
.33 (dd, J ¼ 8.5, 1.7 Hz, 1H), 4.04 (s, 2H).
6
DMSO-d ) d 183.0, 167.9, 151.4, 151.0, 143.3, 141.1, 134.7, 132.4,
4
.3.8 Compound 35. Similar procedures as described for 131.7, 130.4, 123.8, 122.9, 118.2, 111.3, 41.5, 21.3; negative ESI-
1
ꢁ
compound 28. 144 mg (68%) from compound 22 (307 mg). H MS: m/z 399.06 ([M ꢁ H] ) and negative HR-ESI-MS for
ꢁ
NMR (400 MHz, CD
3
OD) d 7.75 (d, J ¼ 1.0 Hz, 1H), 7.51 (d, J ¼
C
22
H
20
N
6
O
2
([M ꢁ H] ) calcd: 399.15640; found: 399.15680.
6
.9 Hz, 1H), 7.38 (dd, J ¼ 6.8, 1.4 Hz, 1H), 4.33 (s, 2H).
4.5.3 Compound 3. Similar procedures as described for
1
4.3.9 Compound 36. Similar procedures as described for compound 1; 43 mg (71%) from compound 30 (55 mg). H NMR
1
compound 28. 76 mg (70%) from compound 23 (167 mg). H (400 MHz, DMSO-d ) d 12.33 (s, 2H), 8.19 (s, 2H), 7.44 (s, 2H),
6
1
3
NMR (400 MHz, CD OD) d 7.07 (dd, J ¼ 8.4, 1.7 Hz, 1H), 6.82 (td, 7.04 (s, 2H), 6.83 (d, J ¼ 7.8 Hz, 2H), 5.00 (s, 4H), 3.79 (s, 6H);
C
3
J ¼ 10.4, 2.0 Hz, 1H), 4.01 (s, 2H).
6
NMR (125 MHz, DMSO-d ) d 183.0, 167.7, 155.8, 154.8, 150.3,
4.3.10 Compound 37. Similar procedures as described for 137.3, 134.9, 128.5, 119.0, 111.6, 110.7, 101.3, 94.7, 55.4, 41.4;
1
ꢁ
compound 28. 64 mg (73%) from compound 24 (140 mg). H negative ESI-MS: m/z 431.01 ([M ꢁ H] ) and negative HR-ESI-MS
ꢁ
NMR (400 MHz, CD
3
OD) d 7.31 (d, J ¼ 8.0 Hz, 1H), 7.17 (ddd, J ¼ for C22
H
20
N
6
O
4
([M ꢁ H] ) calcd: 431.14622; found: 431.14667.
8
.0, 8.0, 4.8 Hz, 1H), 6.93 (dd, J ¼ 10.8, 8.0 Hz, 1H), 4.04 (s, 2H).
4.5.4 Compound 4. Similar procedures as described for
1
4.3.11 Compound 38. Similar procedures as described for compound 1; 33 mg (43%) from compound 31 (66 mg). H NMR
1
compound 28. 54 mg (72%) from compound 25 (110 mg). H (400 MHz, DMSO-d ) d 8.31 (s, 2H), 8.20 (s, 2H), 7.79 (s, 2H), 5.14
6
1
3
NMR (400 MHz, CD OD) d 7.84 (s, 1H), 7.67 (d, J ¼ 8.4 Hz, 1H), (s, 4H); C NMR (125 MHz, DMSO-d ) d 183.2, 168.9, 167.6,
3
6
7
.50 (d, J ¼ 8.5 Hz, 1H), 4.08 (s, 2H).
144.3, 127.0, 125.2, 125.1, 124.0, 123.1, 123.0, 121.1, 114.4 (q, J ¼
4
.3.12 Compound 39. Similar procedures as described for 132.3 Hz), 111.2 (q, J ¼ 120.8 Hz), 45.8; negative ESI-MS: m/z
1
ꢁ
compound 28. 70 mg (72%) from compound 26 (150 mg). H 642.89 ([M ꢁ H] ) and negative HR-ESI-MS for C H F N O
2
4
12 12 6 2
ꢁ
NMR (400 MHz, CD
3
OD) d 7.45 (t, J ¼ 8.8 Hz, 2H), 4.23 (s, 2H). ([M ꢁ H] ) calcd: 643.07463; found: 643.07489.
4.3.13 Compound 40. Similar procedures as described for
4.5.5 Compound 5. Similar procedures as described for
1
1
compound 28. 79 mg (73%) from compound 27 (172 mg). H NMR compound 1; 45 mg (69%) from compound 32 (59 mg). H NMR
(
2
400 MHz, CD OD) d 7.50 (dd, J ¼ 8.8, 4.7 Hz, 1H), 7.15 (dd, J ¼ 9.1, (400 MHz, DMSO-d ) d 8.45 (s, 2H), 8.30 (s, 2H), 8.11 (d, J ¼ 8.8 Hz,
3
6
13
.2 Hz, 1H), 7.00 (ddd, J ¼ 9.3, 9.3, 2.4 Hz, 1H), 4.08 (s, 2H).
2H), 7.72 (d, J ¼ 8.8 Hz, 2H), 5.10 (s, 4H); selected C NMR (125
MHz, DMSO-d ) d 183.1, 168.0, 156.8, 142.6, 117.9, 41.5; negative
6
ꢁ
ESI-MS: m/z 460.99 ([M ꢁ H] ) and negative HR-ESI-MS for
4
.4 Synthesis of compound 41
ꢁ
C H N O ([M ꢁ H] ) calcd: 461.09525; found: 461.09579.
20 14 8 6
A solution of compound 28 (40 mg, 0.27 mmol) and Et
mL) in EtOH (3 mL) was stirred at room temperature for 30 min. compound 1; 30 mg (70%) from compound 33 (39 mg). H NMR
3
N (300
4.5.6 Compound 6. Similar procedures as described for
1
3978 | RSC Adv., 2021, 11, 3972–3980
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400 MHz, DMSO-d
RSC Advances
(
6
) d 12.79 (s, 2H), 8.24 (s, 2H), 7.32–7.20 (m, 2H), 7.34 (d, J ¼ 8.1 Hz, 2H), 7.03 (t, J ¼ 8.7 Hz, 2H), 5.01 (s, 4H);
1
3
13
4H), 5.04 (s, 4H); selected C NMR (100 MHz, DMSO-d ) d 183.5,
C NMR (125 MHz, DMSO-d ) d 183.0, 167.8, 159.6, 157.3,
6
6
ꢁ
1
67.3, 154.5, 146.3 (d, J ¼ 42.8 Hz), 144.0 (d, J ¼ 40.8 Hz), 111.1, 153.0, 110.0, 109.8, 41.1; negative ESI-MS: m/z 407.04 ([M ꢁ H] )
ꢁ
ꢁ
99.4, 41.5; negative ESI-MS: m/z 443.02 ([M ꢁ H] ) and negative and negative HR-ESI-MS for C H F N O ([M ꢁ H] ) calcd:
2
0
14 2 6 2
ꢁ
HR-ESI-MS for C20
found: 443.08801.
H
12
F
4
N
6
O
2
([M ꢁ H] ) calcd: 443.08741; 407.10625; found: 407.10605.
4
.5.7 Compound 7. Similar procedures as described for
4
.6 Synthesis of compound 14
1
compound 1; 52 mg (71%) from compound 34 (69 mg). H NMR
400 MHz, DMSO-d ) d 12.63 (s, 2H), 8.20 (s, 2H), 7.73 (s, 2H),
To a solution of compound 41 (40 mg, 0.15 mmol) in EtOH (2
mL) were added Et N (200 mL) and 1-propylamine (9 mg, 0.16
mmol). The resulting mixture was stirred at room temperature
for 23 h. The formed precipitates were collected through
ltration under reduced pressure and washed with EtOH (20 mL
(
6
3
7
4
1
.49 (d, J ¼ 8.4 Hz, 2H), 7.31 (dd, J ¼ 8.5, 1.7 Hz, 2H), 5.01 (s,
1
3
H); selected C NMR (100 MHz, DMSO-d ) d 182.6, 168.0,
6
ꢁ
53.1, 124.7, 114.2, 41.5; negative ESI-MS: m/z 438.89 ([M ꢁ H] )
ꢁ
and negative HR-ESI-MS for C20
H
14Cl
2
N
6
O
2
([M ꢁ H] ) calcd:
1
ꢀ
5) to give compound 14 (34 mg, 79%) having H NMR (500
4
39.04715; found: 439.04749.
MHz, DMSO-d
6
) d 12.48 (s, 1H), 7.97 (s, 1H), 7.58 (s, 1H), 7.48 (s,
4.5.8 Compound 8. Similar procedures as described for
1
1H), 7.17 (s, 2H), 5.00 (s, 2H), 3.50 (s, 2H), 1.54 (sext, J ¼ 7.1 Hz,
compound 1; 96 mg (73%) from compound 35 (142 mg). H
NMR (400 MHz, DMSO-d ) d 12.62 (s, 2H), 8.22 (s, 2H), 7.60–7.53
1
3
2
H), 0.89 (t, J ¼ 7.3 Hz, 3H); C NMR (125 MHz, DMSO-d )
6
6
1
3
d 183.0, 182.3, 168.4, 167.1, 151.6, 142.9, 134.0, 122.2, 121.4,
(
m, 2H), 7.20 (dd, J ¼ 8.5, 1.6 Hz, 2H), 5.02 (s, 4H); selected
C
118.5, 111.4, 45.1, 41.1, 24.0, 11.2; negative ESI-MS: m/z 283.08
NMR (100 MHz, DMSO-d ) d 183.3, 168.0, 153.1, 126.4, 122.0,
6
ꢁ
ꢁ
ꢁ
([M ꢁ H] ) and negative HR-ESI-MS for C13
H
16
N
2
O
2
([M ꢁ H] )
4
1.5; negative ESI-MS: m/z 526.85 ([M ꢁ H] ) and negative HR-
ꢁ
calcd: 283.11895, found: 283.11703.
ESI-MS for C20
26.94629.
H
14Br
2
N
6
O
2
([M ꢁ H] ) calcd: 526.94612; found:
5
4
.5.9 Compound 9. Similar procedures as described for Conflicts of interest
1
compound 1; 58 mg (72%) from compound 36 (73 mg). H NMR
400 MHz, DMSO-d
) d, 8.19 (s, 2H), 7.22 (dd, J ¼ 8.6, 1.5 Hz,
H), 7.04 (td, J ¼ 10.7, 1.7 Hz, 2H), 5.02 (s, 4H); selected
There are no conicts to declare.
(
2
6
1
3
C
NMR (125 MHz, DMSO-d ) d 183.0, 167.8, 159.1 (d, J ¼ 77.0 Hz),
6
Acknowledgements
1
56.8 (d, J ¼ 55.7 Hz), 153.1, 97.5, 97.3, 97.2, 96.9, 41.3; negative
ꢁ
Financial support from the National Natural Science Founda-
tion of China (No. 21877057) and Jiangmen Program for Inno-
vative Research Team (No. 2018630100180019806) is
acknowledged.
ESI-MS: m/z 442.99 ([M ꢁ H] ) and negative HR-ESI-MS for
ꢁ
C
20
H
12
F
4
N
6
O
2
([M ꢁ H] ) calcd: 443.08741; found: 443.08789.
4
.5.10 Compound 10. Similar procedures as described for
1
compound 1; 51 mg (73%) from compound 37 (61 mg). H NMR
400 MHz, DMSO-d
) d 12.87 (s, 2H), 8.22 (s, 2H), 7.35 (d, J ¼
.6 Hz, 2H), 7.17 (t, J ¼ 8.0 Hz, 1H), 7.16 (t, J ¼ 8.0 Hz, 1H), 6.99
(
7
(
6
Notes and references
1
3
t, J ¼ 8.6 Hz, 2H), 5.05 (s, 4H); C NMR (125 MHz, DMSO-d )
6
d 183.0, 167.7, 152.1, 137.1, 131.2, 122.8, 107.9, 106.8, 106.7,
1 L. Chen, S. N. Berry, X. Wu, E. N. W. Howe and P. A. Gale,
Chem, 2020, 6, 61–141.
2 X.-H. Yu, X.-Q. Hong, Q.-C. Mao and W.-H. Chen, Eur. J. Med.
Chem., 2019, 184, 111782.
ꢁ
4
1.3; negative ESI-MS: m/z 407.01 ([M ꢁ H] ) and negative HR-
ꢁ
ESI-MS for C20
H
14
F
2
N
6
O
2
([M ꢁ H] ) calcd: 407.10625; found:
4
07.10687.
4
.5.11 Compound 11. Similar procedures as described for
3 P. A. Gale, J. T. Davis and R. Quesada, Chem. Soc. Rev., 2017,
46, 2497–2519.
1
compound 1; 42 mg (74%) from compound 38 (52 mg). H NMR
(
7
400 MHz, DMSO-d ) d 12.93 (s, 2H), 8.24 (s, 2H), 7.90 (s, 2H),
4 Z. Li and W.-H. Chen, Mini-Rev. Med. Chem., 2017, 17, 1398–
1405.
5 N. Busschaert, C. Caltagirone, W. Van Rossom and P. A. Gale,
Chem. Rev., 2015, 115, 8038–8155.
6 D. S. Kim and J. L. Sessler, Chem. Soc. Rev., 2015, 44, 532–546.
7 N. Busschaert and A. P. Gale, Angew. Chem., Int. Ed., 2013, 52,
1374–1382.
8 G. W. Gokel and S. Negin, Acc. Chem. Res., 2013, 46, 2824–
2833.
6
1
3
.73 (d, J ¼ 8.3 Hz, 2H), 7.50 (d, J ¼ 8.4 Hz, 2H), 5.08 (s, 4H);
C
NMR (125 MHz, DMSO-d ) d 183.1, 167.9, 154.6, 129.1, 126.4,
1
6
23.7, 122.6 (q, J ¼ 156.1 Hz), 121.0, 118.7, 41.5; negative ESI-
ꢁ
MS: m/z 507.03 ([M ꢁ H] ) and negative HR-ESI-MS for
ꢁ
C
22
H
4
14
F
6
N
6
O
2
([M ꢁ H] ) calcd: 507.09986; found: 507.10004.
.5.12 Compound 12. Similar procedures as described for
1
compound 1; 54 mg (71%) from compound 39 (67 mg). H NMR
(
(
400 MHz, DMSO-d ) d 8.13 (s, 2H), 7.58 (t, J ¼ 9.2 Hz, 4H), 4.99
6
13
s, 4H); selected C NMR (125 MHz, DMSO-d ) d 183.0, 167.8,
9 P. A. Gale, R. P ´e rez-Tom ´a s and R. Quesada, Acc. Chem. Res.,
2013, 46, 2801–2813.
6
1
53.7, 147.8 (d, J ¼ 76.5 Hz), 145.4 (d, J ¼ 75.7 Hz), 41.4; negative
ꢁ
ESI-MS: m/z 442.94 ([M ꢁ H] ) and negative HR-ESI-MS for 10 F. De Riccardis, I. Izzo, D. Montesarchio and P. Tecilla, Acc.
ꢁ
C
20
H
4
12
F
4
N
6
O
2
([M ꢁ H] ) calcd: 443.08741; found: 443.08786.
Chem. Res., 2013, 46, 2781–2790.
.5.13 Compound 13. Similar procedures as described for 11 S. K. Ko, S. K. Kim, A. Share, V. M. Lynch, J. Park,
1
compound 1; 61 mg (72%) from compound 40 (76 mg). H NMR
400 MHz, DMSO-d ) d 12.56 (s, 2H), 8.17 (s, 2H), 7.54–7.51 (m,
W. Namkung, W. V. Rossom, N. Busschaert, P. A. Gale,
J. L. Sessler and I. Shin, Nat. Chem., 2014, 6, 885–892.
(
6
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2021 The Author(s). Published by the Royal Society of Chemistry
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RSC Advances
Paper
1
1
1
1
1
2 C. R. Elie, G. David and A. R. Schmitzer, J. Med. Chem., 2015, 32 N. Busschaert, I. L. Kirby, S. Young, S. J. Coles, P. N. Horton,
5
8, 2358–2366.
M. E. Light and P. A. Gale, Angew. Chem., Int. Ed., 2012, 51,
4426–4430.
33 Z. Li, L.-Q. Deng, Y. Chen, T. Wu and W.-H. Chen, Bioorg.
Med. Chem. Lett., 2016, 26, 3665–3668.
34 C.-C. Peng, Z. Li, L.-Q. Deng, Z.-F. Ke and W.-H. Chen, Bioorg.
Med. Chem. Lett., 2016, 26, 2442–2445.
35 L. Gonzalez-Mendoza, B. Altava, M. I. Burguete,
J. Escorihuela, E. Hernando, S. V. Luis, R. Quesada and
C. Vicent, RSC Adv., 2015, 5, 34415–34423.
3 W.-H. Chen, X.-B. Shao, R. Moellering, C. Wennersten and
S. L. Regen, Bioconjugate Chem., 2006, 17, 1582–1591.
4 T. Saha, A. Gautam, A. Mukherjee, M. Lahiri and P. Talukdar,
J. Am. Chem. Soc., 2016, 138, 16443–16451.
5 T. Saha, M. S. Hossain, D. Saha, M. Lahiri and P. Talukdar, J.
Am. Chem. Soc., 2016, 138, 7558–7567.
6 V. Soto-Cerrato, P. Manuel-Manresa, E. Hernando,
S. Calabuig-Farinas, A. Mart ´ı nez-Romero, V. Fernandez-
Duenas, K. Sahlholm, T. Knopfel, M. Garc ´ı a-Valverde, 36 J. Gravel and A. R. Schmitzer, Supramol. Chem., 2015, 27,
A. M. Rodilla, E. Jantus-Lewintre, R. Farras, F. Ciruela, 364–371.
R. Perez-Tomas and R. Quesada, J. Am. Chem. Soc., 2015, 37 J. Kempf, N. Noujeim and A. R. Schmitzer, RSC Adv., 2014, 4,
37, 15892–15898. 42293–42299.
7 E. Hernando, V. Soto-Cerrato, S. Cortes-Arroyo, R. P ´e rez- 38 C. R. Elie, G. David and A. R. Schmitzer, J. Med. Chem., 2015,
1
1
1
1
Tom ´a s and R. Quesada, Org. Biomol. Chem., 2014, 12,
771–1778.
8 S. J. Moore, M. Wenzel, M. E. Light, R. Morley,
58, 2358–2366.
1
39 C.-R. Elie, M. Charbonneau and A. R. Schmitzer, RSC Med.
Chem., 2012, 3, 1231–1234.
S. J. Bradberry, P. Gomez-Iglesias, V. Soto-Cerrato, R. P ´e rez- 40 C.-R. Elie, N. Noujeim, C. Pardin and A. R. Schmitzer, Chem.
Tom ´a s and P. A. Gale, Chem. Sci., 2012, 3, 2501–2509.
Commun., 2011, 47, 1788–1790.
9 S. J. Moore, C. J. E. Haynes, J. Gonzalez, J. L. Sutton, 41 X.-H. Yu, X.-Q. Hong and W.-H. Chen, Org. Biomol. Chem.,
S. J. Brooks, M. E. Light, J. Herniman, G. J. Langley, 2019, 17, 1558–1571.
V. Soto-Cerrato, R. Perez-Tomas, I. Marques, P. J. Costa, 42 X.-H. Yu, C.-C. Peng, X.-X. Sun and W.-H. Chen, Eur. J. Med.
V. Felix and P. A. Gale, Chem. Sci., 2013, 4, 103–117. Chem., 2018, 152, 115–125.
0 H. Li, H. Valkenier, A. G. Thorne, C. M. Dias, J. A. Cooper, 43 C.-C. Peng, M.-J. Zhang, X.-X. Sun, X.-J. Cai, Y. Chen and
2
M. Kieffer, N. Busschaert, P. A. Gale, D. N. Sheppard and
A. P. Davis, Chem. Sci., 2019, 10, 9663–9672.
1 X.-J. Cai, Z. Li and W.-H. Chen, Mini-Rev. Org. Chem., 2018,
W.-H. Chen, Org. Biomol. Chem., 2016, 14, 8232–8236.
44 C. Montoya, R. Cervantes and J. Tiburcio, Tetrahedron Lett.,
2015, 56, 6177–6182.
2
2
2
2
1
5, 148–156.
45 J. Cai and J. L. Sessler, Chem. Soc. Rev., 2014, 43, 6198–6213.
2 X.-H. Yu, X.-J. Cai, X.-Q. Hong, K. Y. Tam, K. Zhang and 46 M. Lisbjerg, H. Valkenier, B. M. Jessen, H. Al-Kerdi,
W.-H. Chen, Future Med. Chem., 2019, 11, 1091–1106.
3 X. Bao, X. Wu, S. N. Berry, E. N. W. Howe, Y.-T. Chang and
P. A. Gale, Chem. Commun., 2018, 54, 1363–1366.
A. P. Davis and M. Pittelkow, J. Am. Chem. Soc., 2015, 137,
4948–4951.
47 L. Yin, W.-R. Xu, Z.-G. Wang, D.-T. Zhang, J. Jia, Y.-Q. Ge,
Y. Li and J.-W. Wang, Arkivoc, 2010, (9), 196–205.
4 N. Busschaert, S. H. Park, K. H. Beak, Y. P. Choi, J. H. Park,
E. N. W. Howe, J. R. Hiscock, L. E. Karagiannids, I. Marques, 48 M. Bibian, R. J. Rahaim, J. Y. Choi, Y. Noguchi, S. Schurer,
V. Felix, W. Namkung, J. L. Sesseler, P. A. Gale and I. Shin,
Nat. Chem., 2017, 9, 667–675.
5 X.-J. Cai, Z. Li and W.-H. Chen, Bioorg. Med. Chem. Lett.,
W. Chen, S. Nakanishi, K. Licht, L. H. Rosenberg, L. Li,
Y. Feng, M. D. Cameron, D. R. Duckett, J. L. Cleveland and
W. R. Roush, Bioorg. Med. Chem. Lett., 2013, 23, 4374–4380.
49 J. T. Davis, P. A. Gale, O. A. Okunola, P. Prados, J. C. Iglesias-
Sanchez, T. Torroba and R. Quesada, Nat. Chem., 2009, 1,
138–144.
2
2017, 27, 1999–2002.
2
6 X. Wu, L. W. Judd, E. N. W. Howe, A. M. Withecombe,
V. Soto-Cerrato, H. Li, N. Busschaert, H. Valkenier,
R. Perez-Tomas, D. N. Sheppard, Y.-B. Jiang, A. P. Davis 50 S. Bhosale and S. Matile, Chirality, 2006, 18, 849–856.
and P. A. Gale, Chem, 2016, 1, 127–146.
51 S. Dutta, B. G. Watson, S. Mattoo and J.-C. Rochet, Bio-
Protoc., 2020, 10, e3690.
52 S.-P. Zheng, J.-J. Jiang, A. V. D. Lee and M. Barboiu, Angew.
Chem., Int. Ed., 2020, 132, 19082–19088.
53 P. A. Gale, C. C. Tong, C. J. E. Haynes, O. Adeosun,
D. E. Gross, E. Karnas, E. M. Sedenberg, R. Quesada and
J. L. Sessler, J. Am. Chem. Soc., 2010, 132, 3240–3241.
2
2
2
3
7 R. B. P. Elmes, N. Busschaert, D. D. Czech, P. A. Gale and
K. A. Jolliffe, Chem. Commun., 2015, 51, 10107–10110.
8 S. J. Edwards, H. Valkenier, N. Busschaert, P. A. Gale and
A. P. Davis, Angew. Chem., Int. Ed., 2015, 54, 4592–4596.
9 Z. Li, L.-Q. Deng, J.-X. Chen, C.-Q. Zhou and W.-H. Chen, Org.
Biomol. Chem., 2015, 13, 11761–11769.
0 N. Busschaert, R. B. P. Elmes, D. D. Czech, X. Wu, I. L. Kirby, 54 Z. Li, X.-H. Yu, Y. Chen, D.-Q. Yuan and W.-H. Chen, J. Org.
E. M. Peck, K. D. Hendzel, S. K. Shaw, B. Chan, B. D. Smith,
K. A. Jolliffe and P. A. Gale, Chem. Sci., 2014, 5, 3617–3626.
1 L.-Q. Deng, Y.-M. Lu, C.-Q. Zhou, J.-X. Chen, B. Wang and
W.-H. Chen, Bioorg. Med. Chem. Lett., 2014, 24, 2859–2862.
Chem., 2017, 82, 13368–13375.
55 C. J. E. Haynes, N. Busschaert, I. L. Kirby, J. Herniman,
M. E. Light, N. J. Wells, I. Marques, V. F ´e lix and P. A. Gale,
Org. Biomol. Chem., 2014, 12, 62–72.
3
3980 | RSC Adv., 2021, 11, 3972–3980
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