Multi-stimuli controlled release of a transmembrane chloride ion carrier from a sulfonium-linked procarrier

Article abstract of DOI:10.1039/d0ob00938e

In recent times, anion transporters have received substantial consideration due to their ability to disrupt the ionic equilibrium across membrane bilayers. While numerous Cl- ion transporters were developed for channelopathies, unfortunately, poor aqueous solubility precluded their bioapplicability. Herein, we demonstrate the development of a multi-stimuli activatable anion transport approach to induce regulated transport of Cl- ions across membranes under specific conditions. The sulfonium-based procarrier was initially inactive, but the transmembrane transport of Cl- ions was activated in the presence of stimuli such as glutathione (GSH), reactive oxygen species (ROS) and light. The release of the hydrophobic anionophore from the aqueous-soluble procarrier under specific conditions leads to the successful transport of Cl- ions. Under physiological conditions, these anion carriers follow an antiport exchange mechanism to transport Cl- ions across lipid bilayers. Such multi-stimuli activatable procarriers have great potential to combat various types of channelopathies, including cancer, cystic fibrosis, kidney stones, myotonia, and others.

Full text of DOI:10.1039/d0ob00938e

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Multi-stimuli controlled release of a
transmembrane chloride ion carrier from
Cite this: DOI: 10.1039/d0ob00938e
a sulfonium-linked procarrier†
Sribash Das,‡ Oindrila Biswas,‡ Nasim Akhtar, Anjali Patel and Debasis Manna
*
In recent times, anion transporters have received substantial consideration due to their ability to disrupt the
ionic equilibrium across membrane bilayers. While numerous Cl− ion transporters were developed for chan-
nelopathies, unfortunately, poor aqueous solubility precluded their bioapplicability. Herein, we demonstrate
the development of a multi-stimuli activatable anion transport approach to induce regulated transport of
Cl− ions across membranes under specific conditions. The sulfonium-based procarrier was initially inactive,
but the transmembrane transport of Cl− ions was activated in the presence of stimuli such as glutathione
(GSH), reactive oxygen species (ROS) and light. The release of the hydrophobic anionophore from the
aqueous-soluble procarrier under specific conditions leads to the successful transport of Cl− ions. Under
physiological conditions, these anion carriers follow an antiport exchange mechanism to transport Cl− ions
across lipid bilayers. Such multi-stimuli activatable procarriers have great potential to combat various types
of channelopathies, including cancer, cystic fibrosis, kidney stones, myotonia, and others.
Received 6th May 2020,
Accepted 27th October 2020
DOI: 10.1039/d0ob00938e
rsc.li/obc
Lipid-soluble synthetic ionophores catalyze the transport of
ions across hydrophobic membranes to promote apoptosis.
Introduction
Multicellular organisms promote cell proliferation and elimin- These molecules generally do not bind to any target specific
ate potentially harmful cells to maintain tissue homeostasis. proteins or genetic materials. Therefore, it is postulated that
However, disorders in apoptotic pathways are linked to neuro- the synthetic ion transporters can overcome the resistance
degenerative disorders, cancer, and other diseases. Cancer associated with the overexpression and mutations of genes
cells evade apoptosis even though they are under persistent and proteins. However, the lipophilicity of these synthetic ion
stress stimuli such as oxidative stress and cellular hypoxia.1–5 carriers precluded their target specific cellular delivery and
The oxidative stress allows the overproduction of reactive in vivo applications. Poor aqueous solubility is one of the
oxygen species (ROS) in the cell, which leads to mitochondrial reasons for the clinical trial failure of obatoclax, a Cl− ion
dysfunction. Cancer cells also produce excess antioxidants carrier and inhibitor of the Bcl-2 family of antiapoptotic
such as glutathione (GSH) to minimize the effect of ROS and proteins.19–21 Therefore, an additional delivery system is
prevent oxidative stress-mediated apoptosis. However, the over- required to accomplish the expected biological effects of these
production of GSH also allows cancer cells to develop drug re- lipophilic ion carriers. Hence, a novel strategy is needed to
sistance.6 Therefore, the development of a new strategy that mitigate the challenges of selective cellular uptake and target
targets these overproduced ROS and GSH to induce apoptosis specific deliverability of hydrophobic anionophores.
in cancer cells is highly beneficial.
Previously, we developed a stimuli-responsive Cl− ion trans-
Recent studies revealed that the apoptosis of cancer cells is port strategy for promoting controlled transport of Cl− ions
also associated with the homeostasis of several ions, including across lipid bilayers. We were successful in regenerating the
potassium and chloride. In this regard, synthetic ionophores lipophilic anionophores from water-soluble proanionophores
have been developed as potential anticancer agents.4,5,7–18 after in situ cleavages of the hydrophilic accessories in the
presence of glutathione (GSH).19 Recently, this approach has
been used for constructing ion-channels to induce cancer cell
apoptosis. However, the poor aqueous solubility of the 2-hydro-
xyisophthalamide-based aninophore might be the main
Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781039,
India. E-mail: dmanna@iitg.ac.in
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
obstacle for its deliverability at the specific site and in vivo
d0ob00938e
applications.22 Previously, a nitrobenzyl-based cationic linker
‡These authors contributed equally to this work.
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was also used for improving the aqueous solubility of the
hydrophobic estrogen receptor antagonist, tamoxifen.23 Hence,
stimuli-mediated alteration of the solubility of the ion trans-
porters can be considered as one of the promising therapeutic
approaches in cancer.
Herein, we introduce a GSH-, ROS-, and photo-responsive
anion transport strategy to regenerate lipophilic anionophores
from water-soluble proanionophores and promote controlled
transport of Cl− ions across lipid bilayers. The use of only
external stimuli, such as light, has several limitations as it has
lower tissue penetration ability.24 The smaller change in the
pH gradient between cancerous and normal cells may not be
adequate for obtaining reliable in vivo results.10,11,15 We
hypothesize that this multi-stimuli-responsive anion transport
strategy could be beneficial for targeting different types of
cancer cells. The higher intracellular levels of GSH and ROS in
cancer cells in comparison with those in normal healthy cells
would preferentially release anionophores within the cancer
cells, leading to the induction of apoptosis by perturbing the
ion homeostasis. The GSH-mediated cleavage of sulfonium
moieties is used in several applications, including drug deliv-
ery and selective protein and peptide modifications.25–27 The
installation of a sulfonium moiety also improves the viabilities
and cellular uptake compared to other onium-based moieties
and leads to facile reductive cleavage under physiological con-
ditions.25 The sulfonamide containing dipodal compounds
showed chloride-mediated apoptosis-inducing activities in
cancer cells.8 Recently, we demonstrated the development of
S-methylcysteamine-based thiourea containing dipodal com-
pounds. However, its aggregation properties restricted its
further applications.19 We hypothesized that the installation of
a 4-carboxy-2-nitrobenzyl sulfonium moiety would increase the
aqueous solubility of the corresponding anionophore and
allow its removal at high GSH and ROS concentrations or in
the presence of light (Fig. 1A). Therefore, we installed a
4-carboxy-2-nitrobenzyl group linked via the sulfonium moiety,
as the group is responsive to its linking to membrane-active
anionophores. Substituted aryl groups were attached at the
other end of the 1,3-phenylenedimethanamine scaffold to opti-
mize the ion transport efficiency. The outcome of our studies
revealed that the water-soluble proanionophores successfully
release the membrane active ionophores in the presence of
stimuli such as GSH, ROS, and light, which transport Cl− ions
across the model lipid bilayers.
Fig. 1 Schematic diagram representing the stimuli-responsive release
of an anionophore from its proanionophore (A). Synthesis of aniono-
phores and proanionophores (B).
rate water-soluble proanionophores. All the synthesized com-
pounds were purified by column chromatography and charac-
terized by 1H NMR, 13C NMR, and HRMS analysis.
1
The H NMR titration experiment was conducted to investi-
gate the Cl− ion recognition competence of the
compounds.7–10,19,22 Tetrabutylammonium chloride (TBACl)
was used as the source of Cl− ions. The downfield chemical
shifts of N–H signals suggest the interaction of Cl− ions with
the compounds through hydrogen bonding. The strength of
interactions was scrutinized by calculating the binding
affinities (Ka) from the chemical shift (Δδ) values for the N–H
signals, using the BindFit v0.5 program. The inbuilt 1 : 1
binding model provided the best fit for the titration curves.
The calculated Ka values revealed that compounds 1b and 1c
have higher Cl− ion binding capabilities (Table
1 and
Fig. S1–4†). The 1H NMR titration experiment was also per-
formed in CD3CN solvent to investigate the effect of the
solvent on the Cl− ion recognition properties of the com-
pounds. The calculated Ka values of compound 1c were 69.08
(Fig. S5†) and 105.21 M−1 (Fig. S3†) in DMSO-d6 and CD3CN
Table 1 Physical properties of the thiourea derivatives
Results and discussion
Compound
Clog Pa
Kab (M−1
)
EC50c (µM) (n)
Compounds 1a–1d were synthesized from 1-(3-(isothiocyanato-
methyl)benzyl)-3-(2-(methylthio)ethyl)thiourea via conden-
sation with the corresponding amines. The 4-carboxy-2-nitro-
benzyl sulfonium appended compound 2 was synthesized by
reacting compound 1c with 4-bromomethyl-3-nitrobenzoic
acid (Fig. 1B). Several thiourea-based dipodal ionophores have
already been described,8,19 but the presence of a methyl-
thioethyl moiety allows these synthesized compounds to gene-
1a
1b
1c
1d
4.18
5.07
5.95
3.93
32.07 0.31
49.51 0.27
69.08 0.40
24.58 0.37
3.66 0.36 (0.59)
0.08 0.01 (0.63)
0.04 0.02 (1.04)
3.35 0.69 (0.81)
a Clog P: Log P values were computed using the Marvin Sketch 17.27
program. b Binding constant (Ka) values were calculated by monitoring
the changes in chemical shifts of both N–Ha and N–Hb protons using
the BindFit v0.5 program. c EC50 values were calculated using the
modified Hill equation, n = Hill coefficient.
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solvent, respectively. The difference in Ka values of compound = 7.8) across EYPC/CHOL-LUV . HPTS.7–10,19,22 A substantial
1c could be due to the competition of DMSO with TBACl in variance in the extent of anion transport abilities of compound
Cl− ion recognition.
1c (Fig. 2B) was observed when the ion transport activity was
The ion transport activities of compounds 1a–1d and 2 measured with intravesicular NaCl and an iso-osmolar extrave-
were evaluated by fluorescence assay using model large unila- sicular NaX salt (Cl−, Br−, I−, F−, and SCN−). Although the use
mellar vesicles (LUVs). The pH-sensitive ratiometric fluoro- of intravesicular NaCl and an iso-osmolar extravesicular M+/
phore, 8-hydroxypyrene-1,3,6-trisulfonate (HPTS, pKa = 7.3), Cl− salt (M+ = Li+, Na+, K+, Rb+, and Cs+) led to comparable Cl−
was encapsulated within the LUVs of egg-yolk phosphatidyl- ion transport efficiencies, their pattern of Cl− ion transport
choline (EYPC) and cholesterol (CHOL) lipids with an internal properties could be different (Fig. S10†). The outcome of the
and external pH value of 7.2. The transmembrane ion trans- ion transport selectivity experiments revealed that the com-
port activities of the compounds were measured by applying a pound has Cl− ion transport selectivity over the other investi-
pH gradient with the addition of NaOH in the extravesicular gated anions. However, the tested metal cations may not play
buffer (pHin = 7.2, pHout = 7.8).7–10,19,22 The alteration of any significant role in the ion transport process.
internal pH in the presence of compounds was examined by
The HPTS assay in the absence and presence of 4-(trifluoro-
the change in the fluorescence intensity of HPTS at 510 nm methoxy)phenylhydrazone (FCCP; a protonophore) and valino-
(λex = 450 nm). The initial screening revealed that compounds mycin (a selective K+ transporter) was also performed to obtain
1b and 1c efficiently transported Cl− ions across the lipid direct experimental evidence of the preferential Cl− ion trans-
bilayer (Fig. 2A). Compounds 1a–1d showed very similar Cl− port mechanism of the potent compound.7–10,19,22 An increase
ion binding capabilities in DMSO-d6 solvent but compounds in the Cl− ion transport rate in the presence of FCCP suggests
1b and 1c showed higher Cl− ion transport abilities compared a cooperative effect between the compound and FCCP, elimi-
to 1a and 1d, which could be due to the difference in their nating the possibility of H+ ion transport by the compound
lipophilicity (log P values <5 for compounds 1a and 1d), which (Fig. 3A). Similarly, the HPTS assay was also performed in the
is considered as one of the essential criteria for their effective absence and presence of valinomycin. The valinomycin-
permeability across biological membranes and transmem- mediated transport of K+ ions from the extravesicular to intra-
brane Cl− ion transport ability (Table 1). The poor Cl− ion vesicular solution could induce the efflux of Cl− ions and/or
transport efficiency of the sulfonium-based compound 2 could influx of OH− ions to maintain the pH gradient. A substantial
be due to its higher aqueous solubility. The dose-dependent improvement in the Cl− ion transport in the presence of vali-
HPTS assay showed that compounds 1b and 1c have over 45- nomycin suggests the OH−/Cl− antiport mechanism (Fig. 3B).
and 88-fold higher Cl− ion transport efficacy than compounds
1a and 1d, respectively, under similar experimental conditions
(Fig. S6–9†). The Hill analysis revealed that the effective con-
centration at 50% (EC50) Cl− ion transport activity was 79.45
and 37.98 nM for compounds 1b and 1c, respectively (Table 1).
Accordingly, the HPTS assay revealed that compound 1c has
higher Cl− ion transport ability than the other synthesized
compounds. Consequently, further biophysical studies were
carried out with compound 1c and its derivatives.
To examine the role of other anions or cations in the Cl−
ion transport activity of compound 1c, the HPTS assay was
conducted in the presence of a pH gradient (pHin = 7.2, pHout
Fig. 3 Assessment of the ion transport activity of compound 1c
(0.038 µM) in the absence and presence of FCCP (4 μM) (A) and valino-
mycin (Val; 2.5 pM) (B). A pH gradient (pHin = 7.2, pHout = 7.8) was
applied to initiate the transport of Cl− ions. The effect of the cholesterol
concentration on the transport of Cl− ions in the presence of compound
1c (0.038 µM) (C). The molar ratios of EYPC and cholesterol were varied
as 6 : 4, 8 : 2, and 10 : 0 to study the effect of cholesterol on the Cl− ion
transport activity of the compounds. The Cl− ion transport efficacy of
compound 1c (2 mM) was recorded across a U-tube by utilizing the Cl−
ion gradient, using a chloride ion-selective electrode (D).
Fig. 2 Transmembrane Cl− ion transport activity of the compounds
(1a–d and 2) across EYPC/CHOL-LUV . HPTS by applying a pH gradient
(A; pHi = 7.2 and pHout = 7.8). The compound concentration was
0.038 µM. Anion transport selectivity of compound 1c (B) under similar
experimental conditions.
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The cooperative effect of compound 1c and valinomycin tion of proaniononophore 2 (1 mmol) was performed in the
suggests the preferential transport of OH− ions over Cl− ions. presence of Fenton’s reagent (1 mM Fe2+ and 5 mM H2O2) in
In the meantime, the cholesterol concentration-dependent 10 mM PBS (pH 7.4) at 37 °C. The time-dependent dealkyla-
transport (Fig. 3C) and U-tube assay (Fig. 3D) established that tion of proaniononophore 2 and the regeneration of the active
compound 1c transports Cl− ions via a carrier pathway.
hydrophobic carrier 1c along with intermediate 4 in the pres-
The stimuli-mediated dealkylation ability of proanionono- ence of Fenton’s reagent were analyzed by HPLC. HRMS ana-
phore 2 was investigated by HPLC and NMR analyses in the lyses further confirmed the generation of intermediates 3 and
absence and presence of GSH, ROS, and light. The GSH and 4 and the active anionophore 1c (Fig. S12 and 13†). The photo-
ROS mediated dealkylation of water-soluble proaniononophore lytic conversion of proaniononophore 2 to the active carrier 1c
1
2, and the in situ production of the active ion carrier 1c was was investigated by H NMR in DMSO-d6 solvent (Fig. 4D and
investigated by HPLC analysis (Fig. 4A–C). The water solubility E). The solution was irradiated using LED lamps (365 nm
of compound 2 was 2.8 mg mL−1. However, to avoid any wavelength) for 10 min. The presence of a new signal at δ =
solvent effect on the transport efficiency, all compounds 10.6 ppm that corresponds to the aldehyde proton indicates
(including compound 2) were initially dissolved in DMSO and the release of 4-formyl-3-nitrosobenzoic acid and regeneration
then diluted in buffer (so that the final DMSO concentration is of compound 1c from 2. The photolytic generation of 4-formyl-
less than 3% (v/v)). The dealkylation of proaniononophore 2 3-nitrosobenzoic acid was also confirmed by FT-IR and HRMS
(1 mmol) was performed in the presence of GSH (2 mM) in analyses (Fig. S14 and 15†).
10 mM PBS (pH 7.4) at 37 °C. HPLC analysis revealed the
The successful regeneration of the active lipophilic carrier
time-dependent dealkylation of proaniononophore 2 and the 1c from water-soluble proaniononophore 2 in the presence of
regeneration of the active hydrophobic carrier 1c along with GSH, ROS, and light encouraged us to study stimuli-activated
intermediate 3 in the presence of GSH. Similarly, the dealkyla- transmembrane ion transport efficiency.19 The time-dependent
improvement of the Cl− ion transport ability also proved the
generation of the active carrier 1c in the presence of GSH,
ROS, and light (Fig. 5). Upon photo-irradiation, the rate of
dealkylation was quite faster in comparison with those in the
presence of GSH and ROS, which is advantageous for their
bio-applicability. The photo-activated strategy can be applied
for only surface phenomena due to the lack of tissue pene-
tration ability of light. The higher levels of intracellular GSH
and ROS in cancer cells could be sufficient enough to release
the active carrier and promote ion transport mediated apopto-
Fig. 5 Chloride ion transport measurement of the Fenton’s reagent (A),
Fig. 4 Proposed mechanisms of the ROS and GSH mediated cleavage
of proanionophore 2 (A). HPLC traces at different time intervals in the
absence and presence of Fenton’s reagent (B) and GSH (C). Proposed
mechanism of the photo-mediated cleavage of proanionophore 2 (D).
1H NMR spectra of proanionophore 2 at different time intervals (E).
glutathione (B) and light (365 nm) (C) mediated release of the active
transporter 1c from proanionophore in the presence of LUVs.
Schematic diagram representing the stimuli-mediated regeneration of
the active anionophore from its proanionophore and its transmembrane
anion transport properties (D).
2
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sis. However, the structural integrity of proanionophore 2 was was monitored by TLC analysis. After the completion of the
intact in the absence of GSH, ROS, and light.
reaction, the mixture was concentrate under reduced pressure.
Then the crude reaction mixture was purified through column
chromatography with a solvent gradient system using ethyl
acetate/hexane (0–10%) to give the target compound as a white
solid (90% yield). The compound was characterized by 1H
Conclusions
Herein, we demonstrated a multistimuli-responsive approach NMR and 13C NMR analysis. 1H NMR (600 MHz, CDCl3): δ
to regenerate an active lipophilic ionophore from a water- 7.32–7.28 (m, 1H), 7.24–7.22 (m, 1H), 7.19–7.17 (m, 2H), 4.64
soluble proanionophore. The sulfonium-based proaniono- (m, 2H), 4.57 (s, 2H), 3.67–3.62 (m, 2H), 2.63–2.60 (m, 2H),
phore was successfully cleaved in the presence of stimuli such 1.98 (s, 3H); 13C NMR (151 MHz, CDCl3): δ 182.2, 135.0, 129.6,
as ROS, GSH, and light. 1H NMR titration experiments con- 126.4, 126.0, 48.6, 47.8, 42.7, 33.3, 14.9.
firmed the binding of the dithiourea derivative to the Cl− ion.
Synthesis of bis(thiourea) derivatives. To a stirring solution
The fluorescence-based assay confirmed that the dithiourea of 1-(3-(isothiocyanatomethyl)benzyl)-3-(2-(methylthio)ethyl)
derivative selectively transports Cl− ions across the lipid thiourea (200 mg, 0.64 mmol) in dry DMF, the corresponding
bilayer. The U-tube and cholesterol concentration-dependent aromatic amine (0.77 mmol) and triethylamine (89 mL,
assays suggest the transport of Cl− ions via a carrier pathway. 0.64 mmol) were added at room temperature under a N2 atmo-
The FCCP and valinomycin assays showed that the Cl−/OH− sphere. Then, the reaction mixture was stirred for 16–22 h at
antiport mechanism is the operative pathway for Cl− ion trans- room temperature, and the reaction was monitored by TLC
port. The regenerated active carrier also successfully transports analysis. After the completion of the reaction, the DMF was
Cl− ions under similar experimental conditions. Overall, this removed through ethyl acetate/ice water workup (minimum
investigation revealed that the multistimuli-responsive strategy five times) using a 100 mL separating funnel. The collected
could be applied for the treatment of ion transport associated organic layers were dried over anhydrous Na2SO4, filtered, and
diseases.
concentrated under reduced pressure. The crude reaction
mixture was purified through column chromatography with a
solvent gradient system using ethyl acetate/hexane (0–10%) to
give the corresponding yellowish compound. For compounds
1a, 1b, 1c, and 1d, the yields were 90%, 85%, 80%, and 95%
respectively.
Experimental section
Synthesis of compounds
Synthesis of 1,3-bis(isothiocyanatomethyl)benzene. The
Synthesis of the sulfonium derivative (2). To a stirring solu-
compound 1,3-bis(isothiocyanatomethyl)benzene was syn- tion of compound 1c (200 mg, 0.37 mmol) in dry CH3CN was
thesized according to the literature procedure.19 In brief, to a added
4-bromomethyl-3-nitrobenzoic acid (106 mg,
stirring solution of 1,3-phenylenedimethanamine (500 mg, 0.41 mmol) at room temperature under a N2 atmosphere.
3.67 mmol) and N,N‘-dicyclohexylcarbodiimide (DCC; 1.5 g) in Then, the reaction mixture was stirred for 24 h under dark con-
dry THF, CS2 (2.3 mL, 36 mmol) was added dropwise at room ditions. The progress of the reaction was monitored by TLC
temperature under a N2 atmosphere. The reaction mixture was analysis. After the completion of the reaction, the solvent was
allowed to stir for 30 min at room temperature, and the pro- removed under reduced pressure. The crude product was puri-
gress of the reaction was monitored by thin-layer chromato- fied by preparative HPLC using the solvent system of water/
graphy (TLC) analysis. After this, the reaction mixture was fil- methanol (0–95%) to give the yellowish solid compound 2
tered through filter paper. The filtrate was concentrated under (70% yield).
reduced pressure. Then the crude reaction mixture was puri-
fied through column chromatography with a solvent gradient
system using ethyl acetate/hexane (0–10%) to furnish the
Characterization of the synthesized compounds
target compound as a yellowish liquid (70% yield). The com-
1-(3-((3-(2-(Methylthio)ethyl)thioureido)methyl)benzyl)-
pound was characterized by 1H NMR and 13C NMR analysis. 3-phenylthiourea (1a). The general procedure as mentioned
1H NMR (600 MHz, CDCl3): δ 7.34–7.30 (m, 1H), 7.21–7.19 (m, in the earlier section was followed, using 1-(3-(isothiocyanato-
2H), 7.15 (s, 1H), 4.64 (s, 4H); 13C NMR (151 MHz, CDCl3): δ methyl)benzyl)-3-(2-(methylthio)ethyl)thiourea
(200
mg,
129.9, 127.6, 124.4, 121.6, 120.0, 43.2.
0.64 mmol), aniline (71.15 mg, 0.77 mmol) and triethylamine
Synthesis of 1-(3-(isothiocyanatomethyl)benzyl)-3-(2-(methyl- (89 mL, 0.64 mmol). The yellowish compound was isolated in
thio)ethyl)thiourea. The compound 1-(3-(isothiocyanato- 90% yield. The compound was characterized by HRMS (ESI),
methyl)benzyl)-3-(2-(methylthio)ethyl)thiourea was synthesized 1H NMR and 13C NMR analysis. 1H NMR (600 MHz, DMSO-d6):
according to the literature procedure.19 In brief, to a stirring δ 9.61 (s, 1H), 8.14 (s, 1H), 7.98 (s, 1H), 7.60 (s, 1H), 7.74–7.43
solution of 1,3-bis(isothiocyanatomethyl)benzene (500 mg, (m, 2H), 7.35–7.32 (m, 2H), 7.31–7.29 (m, 1H), 7.25 (s, 1H),
2.27 mmol) in dry CH3CN was added 2-(methylthio)ethan-1- 7.23–7.22 (m, 1H), 7.19–7.17 (m, 1H), 7.14–7.11 (m, 1H),
amine (186.2 mg, 2.04 mmol) at room temperature under a N2 4.73–4.66 (m, 4H), 3.61 (m, 2H), 2.64–2.62 (t, J = 7.0 Hz, 2H),
atmosphere. Then the reaction mixture was allowed to stir for 2.08 (s, 3H); 13C NMR (151 MHz, CDCl3): δ 181.3, 139.6, 139.4,
5–6 h at room temperature, and the progress of the reaction 129.4, 128.7, 126.8, 126.5, 126.4, 124.8, 123.8, 47.8, 43.3, 32.93,
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15.1; HRMS (ESI) m/z: calculated for C19H24N4S3 (M + H)+: 0.37 mmol) and 4-bromomethyl-3-nitrobenzoic acid (106 mg,
405.1236, found: 405.1236.
0.41 mmol). The crude compound was isolated in 70% yield.
1
1-(3-((3-(2-(Methylthio)ethyl)thioureido)methyl)benzyl)- The compound was characterized by ES-MS, H NMR and 13C
3-(4-(trifluoromethyl)phenyl)thiourea (1b). The general NMR analysis. 1H NMR (600 MHz, DMSO-d6): δ 11.15–11.08
procedure as mentioned in the earlier section was followed, (m, 1H), 9.13–9.07 (m, 1H), 8.44 (s, 1H), 8.39–8.37 (m, 2H),
using
1-(3-(isothiocyanatomethyl)benzyl)-3-(2-(methylthio) 8.14–8.10 (m, 1H), 7.74–7.70 (m, 1H), 7.51 (s, 1H), 7.36–7.27
ethyl)thiourea (200 mg, 0.64 mmol), 4-(trifluoromethyl)aniline (m, 3H), 7.17 (s, 1H), 7.08 (s, 1H), 6.96 (s, 1H), 4.87–4.84 (m,
(124.07 mg, 0.77 mmol) and triethylamine (89 mL, 2H), 4.79–4.75 (m, 2H), 4.61–4.56 (m, 2H), 3.62–3.54 (m, 2H),
0.64 mmol). The crude yellowish compound was isolated in 2.69 (m, 2H), 2.06 (s, 3H); 13C NMR (151 MHz, CDCl3): δ 181.4,
85% yield. The compound was characterized by the HRMS 165.7, 163.6, 148.2, 142.6, 134.6, 133.2, 132.6, 130.6, 129.0,
(ESI), 1H NMR and 13C NMR analysis. 1H NMR (600 MHz, 127.1, 126.3, 126.0, 124.6, 123.1, 122.8, 121.5, 116.4, 47.0, 46.2,
DMSO-d6): δ 9.97 (s, 1H), 8.47 (s, 1H), 8.00 (s, 1H), 7.75–7.73 43.7, 32.1, 31.6, 15.2; HRMS (ESI) m/z: calculated for
(m, 2H), 7.68–7.66 (m, 2H), 7.6 (s, 1H), 7.33–7.30 (m, 1H), 7.26 C29H28F6N5O4S3+ (M): 720.1280, found: 720.1249.
(s, 1H), 7.24–7.23 (m, 1H), 7.20–7.19 (m, 1H), 4.74–4.67 (m, 4
Measurement of anion recognition, anion transport, and
regeneration activities
H), 3.61 (m, 2H), 2.63–2.61 (t, J = 7.0 Hz, 2H), 2.08 (s, 3H); 13C
NMR (151 MHz, CDCl3): δ 182.4, 181.0, 140.8, 137.9, 129.1,
126.8, 126.7, 126.0, 124.8, 122.9, 122.5, 121.2, 48.3, 48.1, 42.9, The detailed experimental procedures for anion recognition,
32.9, 14.9; HRMS (ESI) m/z: calculated for C20H23F3N4S3 (M + chloride ion transport, U-tube assay, vesicle leakage, and other
H)+: 473.1110, found: 473.1110.
biophysical assays are described in the ESI.†
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-((3-(2-(methylthio)-
ethyl)thioureido)methyl)benzyl)thiourea (1c). The general
procedure as mentioned in the earlier section was followed, Conflicts of interest
using
1-(3-(isothiocyanatomethyl)benzyl)-3-(2-(methylthio)
There are no conflicts to declare.
ethyl)thiourea (200 mg, 0.64 mmol), 3,5-bis(trifluoromethyl)
aniline (416.26 mg, 0.77 mmol) and triethylamine (89 mL,
0.64 mmol). The crude yellowish compound was isolated in
1
Acknowledgements
90% yield. The compound was characterized by the ES-MS, H
NMR and 13C NMR analysis. H NMR (600 MHz, DMSO-d6): δ
1
The authors gratefully acknowledge the Department of
Biotechnology, Government of India (MED/2015/04) and the
Science and Engineering Research Board, Government of India
(EMR/2016/005008) for financial support.
10.17 (s, 1H), 8.67 (s, 1H), 8.28 (s, 2H), 7.98 (s, 1H), 7.76 (s,
1H), 7.60 (s, 1H), 7.56–7.31 (t, J = 7.6 Hz, 1H), 7.26 (s, 1H),
7.25–7.23 (m, 1H), 7.20–7.19 (m, 1H), 4.77–4.66 (m, 4H), 3.61
(m, 2H), 2.63–2.61 (t, 2H), 2.07 (s, 3H); 13C NMR (151 MHz,
CDCl3): δ 181.3, 139.8, 131.8, 131.6, 129.1, 126.9, 126.8, 123.8,
122.0, 121.9, 120.2, 118.1, 50.9, 48.2, 42.9, 32.9, 14.6; HRMS
(ESI) m/z: calculated for C29H22F6N4O4S3 (M + H)+: 541.0984,
found: 541.0961.
1-(4-Methoxyphenyl)-3-(3-((3-(2-(methylthio)ethyl)thiour-
eido)methyl)benzyl)thiourea (1d). The general procedure as
mentioned in the earlier section was followed, using 1-(3-(iso-
thiocyanatomethyl)benzyl)-3-(2-(methylthio)ethyl)thiourea
(200 mg, 0.64 mmol), 4-methoxyaniline (94.89 mg, 0.77 mmol)
and triethylamine (89 mL, 0.64 mmol). The crude yellowish
compound was isolated in 95% yield. The compound was
characterized by the ES-MS, 1H NMR and 13C NMR analysis.
1H NMR (600 MHz, DMSO-d6): δ 9.46 (s, 1H), 8.01 (s, 1H), 7.96
(s, 1H), 7.62 (s, 1H), 7.30–7.28 (m, 1H), 7.26–7.24 (m, 2H), 7.22
(s, 1H), 7.20–7.19 (m, 1H), 7.17–7.16 (m, 1H), 6.92–6.90 (m,
2H), 4.70–4.69 (m, 4H), 3.74 (s, 3H), 2.64–2.62 (m, 2H), 2.08 (s,
3H); 13C NMR (151 MHz, CDCl3): δ 182.3, 181.5, 158.9, 138.3,
137.8, 129.6, 128.4, 127.6, 126.9, 126.7, 126.0, 115.3, 55.6, 48.8,
48.1, 42.9, 33.3, 14.9; HRMS (ESI) m/z: calculated for
C20H26N4OS3 (M + H)+: 435.1342, found: 435.1316.
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