Research Article

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Synthesis and biological evaluation of

aza-crown ether–squaramide conjugates as
anion/cation symporters
Xi-Hui Yu‡ ,1 , Xiong-Jie Cai‡ ,1 , Xiao-Qiao Hong‡ ,1 , Kin Yip Tam2 , Kun Zhang3,4 & Wen-Hua
Chen*,1
1
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University,
Guangzhou 510515, PR China
2
Faculty of Health Sciences, University of Macau, Taipa, Macau, PR China
3
School of Biotechnology & Health Sciences, Wuyi University, Jiangmen 529020, PR China
4
International Healthcare Innovation Institute, Jiangmen 529040, PR China
*Author for correspondence: whchen@smu.edu.cn
‡
Authors contributed equally

Aim: Anion/cation symport across cellular membranes may lead to cell apoptosis and be developed as
a strategy for new anticancer drug discovery. Methodology: Four aza-crown ether–squaramide conju-
gates were synthesized and characterized. Their anion recognition, anion/cation symport, cytotoxicity
and probable mechanism of action were investigated in details. Conclusion: These conjugates are able to
form ion-pairing complexes with chloride anions and facilitate the transmembrane transport of anions via
an anion/cation symport process. They can disrupt the cellular homeostasis of chloride anions and sodium
cations and induce the basification of acidic organelles in live cells. These conjugates exhibit moderate
cytotoxicity toward the tested cancer cells and trigger cell apoptosis by mediating the influx of chloride
anions and sodium cations into live cells.

Graphical abstract:

O O
N Cation N
O O
O NH HN O
Anion
O NH HN O
F3C CF3
Control 50 µm Compound
CF3 F3C Alkalization of acidic organelles

Compound
Cation
Anion

Control 25 µm Compound
Anion/cation symport Morphological changes of nucleus

First draft submitted: 19 December 2018; Accepted for publication: 6 February 2019; Published online:
8 July 2019

Keywords: anion/cation symporter • anionophoric activity • antiproliferative activity • aza-crown ether • squaramide

10.4155/fmc-2018-0595 
C 2019 Newlands Press Future Med. Chem. (2019) 11(10), 1091–1106 ISSN 1756-8919 1091
Research Article Yu, Cai, Hong, Tam, Zhang & Chen

Facilitated transmembrane transport of anions across biomembranes is a ubiquitous cellular process and plays a
crucial role in maintaining the ionic homeostasis of cells [1]. It is well known that the transport of anions across
cellular membranes may be accomplished via several modes of action by transmembrane anion channels or carriers,
including anion exchange and anion/cation symport. Because anion/cation symport is closely associated with
some ‘channelopathies’, such as Bartter syndrome [2], Gitelman syndrome [3], deafness [4], renal tubular acidosis [5]
and thyroid diseases [6], considerable interests have been attracted in developing potent anion/cation symporters
with potential bioactivity [7–15]. In 2003 Smith et al. have reported the first example that macrocyclic receptors
based on aza-crown ethers and isophthalamides are able to facilitate the transport of NaCl or KCl across vesicular
membranes [16]. In 2014, Jeong et al. have described the synthesis of 18-aza-crown-6 or 15-aza-crown-5-appended,
phenylurea-based carriers that are able to transport alkali metal halides, such as KCl and NaCl, via an M+ /Cl-
symport process across 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes [17]. They
have further shown that the transport activity and selectivity may be regulated by carefully modifying the cation-
and anion-binding sites. Gale et al. have demonstrated that 1,2,3-triazole-strapped calix[4]pyrroles are able to
mediate the selective transport of an ion pair such as RbCl or CsCl [12,13,18]. In general, these anion/cation
symporters possess a cation-binding domain that can assist the transmembrane transport of anions through the
formation of ion pairs. Interestingly, some anion/cation symporters exhibit promising biological activity. Typical
examples include naturally occurring prodigiosin and its synthetic analogs having strong antitumor activity as
H+ /Cl- symporters [19,20]. However, the mechanism of biological action of anion/cation symporters remains to be
further explored. Until recently Gale et al. have shown that pyridine diamide-strapped calix[4]pyrroles are able to
facilitate the coupled transport of chloride anions and sodium cations into live cells, leading to an increase in the
concentrations of intracellular chloride anions and sodium cations and further cell death [21].
As part of our ongoing projects towards the development of novel small-molecule anion transporters with
promising bioactivity [22,23], we have become keenly interested in anion/cation symporters, in particular based
on squaramides [24–27]. It has been reported that squaramides are able to recognize anions and exhibit unique
anionophoric activity as anion exchangers [28–34], such as pH-dependent transport of chloride anions [33,34].
These unique properties of squaramides together with the abovementioned structural features of the anion/cation
symporters reported to date [16–18], led us to infer that incorporating squaramides with a cation-binding functionality,
such as aza-crown ethers that have been shown to function as cation-conducting hydraphiles efficiently [35], would
switch the anion exchange mechanism of action of squaramides into anion/cation symport. Though crown ethers
and squaramides have frequently been used as receptors for cations and anions, respectively, their combination
as ion pair receptors are less common [36–38], in particular in the application as anion/cation symporters. With
this background in mind, herein we designed and synthesized aza-crown ether–squaramide conjugates 1–4 having
3,5-bis(trifluoromethyl)phenyl or 4-trifluoromethyl phenyl substituents (Figure 1) and systematically studied their
anion recognition, transmembrane anion/cation symport, antiproliferative activity toward human solid tumor
cells and the probable mechanism of biological action. Here we chose 3,5-bis(trifluoromethyl)phenyl and 4-
trifluoromethylphenyl groups as the substituents, according to the unique properties of trifluoromethyl groups [39] as
well as the reported structure–activity relationships of arylsquaramides as anion transporters [28,31,34]. In conjugates
1–4, the squaramido units serve as anion-binding domains; whereas, the aza-crown ether moieties serve as cation-
complexing units and are expected to facilitate the transmembrane transport of anions through the formation of
ion pair complexes (Figure 1).

Materials & methods

Generals
NMR spectra were acquired on a Bruker Avance AV 400 spectrometer and the residual deuterium solvents were used
as internal standards. Low resolution (LR) ESI–MS, LR ESI–TOF MS and high resolution (HR) ESI–MS spectra
were measured on Waters UPLC/Quattro Premier XE, SCIEX API-TOF MS5000 and Bruker maXis 4G ESI-Q-
TOF mass spectrometers, respectively. Analytical thin-layer chromatography (TLC) was carried out on silica gel
plates 60 GF254 and detected with UV lamp and iodine staining. An Avanti’s Mini-Extruder (Avanti Polar Lipids,
Inc., AL, USA) was used to prepare the liposomes (100 nm diameter), and extrusion approaches through Nuclepore
track-etched polycarbonate membranes (100 nm, Whatman, NJ, USA) were adopted. The efflux of chloride anions
out of liposomes was detected on a Mettler-Toledo Seven Compact S220 ionometer equipped with a Mettler-Toledo

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 Synthesis & biological evaluation of aza-crown ether–squaramide conjugates as anion/cation symporters Research Article

O O O O O O
R1 R1
N N
R2 N N N N R2
H H O O H H
R1 R1
1: R1 = CF3, R2 = H
2: R1 = H, R2 = CF3
O O
N Cation N
O O O O O O
R1 O NH HN O
N N
R2 N N Anion
H H O O O NH HN O
R1 F3C CF3
3: R1 = CF3, R2 = H
4: R1 = H, R2 = CF3
CF3 F3C

Figure 1. Structures of aza-crown ether–squaramide conjugates 1–4 and the proposed interaction mode of
compound 1 with cations and anions.

PerfectIon™ chloride ion selective electrode. Fluorescence spectra were measured on an LS55 spectrofluorimeter. A
Tecan Infinite M1000 PRO microplate reader was used to measure the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2-H-tetrazolium bromide (MTT)-based cytotoxicity and accomplish the N-(ethoxycarbonylmethyl)-6-methoxy
quinolinium bromide (MQAE) and sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM)
assays. A Carl Zeiss Axio Observer A1 microscope was utilized to explore the acridine orange (AO), Hoechst 33342
and JC-1 staining experiments.

Chemicals
The specific chemicals used in this study were egg-yolk L-α-phosphatidylcholine (EYPC), 8-hydroxypyrene-1,3,6-
trisulfonic acid trisodium salt (HPTS) and SBFI-AM from Sigma Chemical Co. (MO, USA); calcein, cholesterol,
AO and Hoechst 33342 from J&K Chemical Co. (Beijing, China), MTT from Amresco (OH, USA), MQAE from
Aladdin (Shanghai, China) and JC-1 mitochondrial membrane potential detection kit from Genview (FL, USA).
All the other commercially available chemicals and reagents were directly used upon receipt.

Chemistry
The synthesis of compounds 1–4 is outlined in Figure 2. Compounds 7 and 9 were prepared from
1,10-diaza-18-crown-6 using the reported procedures [40,41]. Reaction of 1,10-diaza-18-crown-6 with N-(2-
bromoethyl)phthalimide of different molar ratios gave compound 5 or 6. Alkylation of compound 6 with iodoethane
gave compound 8. Hydrazinolysis of compounds 5 and 8 gave compounds 7 and 9, respectively. Compounds 10
and 11 were prepared according to the reported procedures [25,42]. Finally, compounds 1–4 were synthesized from
the reaction of compounds 7 and 9 with compound 10 or 11 according to the procedures described in litera-
ture [25,42]. The structures of compounds 1–4 were confirmed by using NMR (1 H and 13 C) and ESI–MS (LR and
HR).

Synthesis of compound 5
A mixture of 1,10-diaza-18-crown-6 (100 mg, 0.38 mmol), N-(2-bromoethyl)phthalimide (291 mg, 1.14 mmol)
and anhydrous Na2 CO3 (121 mg, 1.14 mmol) in CH3 CN (5.0 ml) was heated under reflux for 18 h. After the
mixture was cooled to room temperature and filtered, the solvent was removed under reduced pressure. Purification
by column chromatography (SiO2 , CH2 Cl2 /CH3 OH, 40/1–30/1, v/v) afforded compound 5 (135 mg, 59%)
having 1 H NMR (CDCl3 , 400 MHz) δ 7.84–7.81 (m, 4H), 7.72–7.69 (m, 4H), 3.75 (t, J = 6.8 Hz, 4H), 3.50 (t,
J = 5.6 Hz, 8H), 3.45 (br, 8H), 2.82–2.78 (m, 12H) and ESI–MS: m/z 609.36 ([M+H]+ ), 631.30 ([M+Na]+ ).
The 1 H NMR data were in agreement with the ones reported in literature [41].

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Research Article Yu, Cai, Hong, Tam, Zhang & Chen

O O O O O O O O
(i) (iii)
NH HN N N N N H2N N N NH2

O O O O O O O O (v)
1: R1 = CF3, R2 = H
2: R1 = H, R2 = CF3
5 7
O O
O O R1
(iv)
i) R2 N O
O H
O R1

10: R1 = CF3, R2 = H
11: R1 = H, R2 = CF3 (v) 3: R1 = CF3, R2 = H
4: R1 = H, R2 = CF3
O O O O O O O O
(ii) (iii)
NH N N N N N N N NH2
O O O O O O O O

6 8 9

Figure 2. Synthesis of compounds 1–4. Reagents and conditions: (i) N-(2-bromoethyl)phthalimide, anhydrous Na2 CO3 , CH3 CN, reflux; (ii)
CH3 CH2 I, CH3 CN, reflux; (iii) (a) NH2 NH2 · H2 O, EtOH, (b) HCl and (c) LiOH; (iv) 3,5-bis(trifluoromethyl)aniline or 4-trifluoromethylaniline,
Zn(CF3 SO3 )2 , anhydrous EtOH, room temperature; (v) Et3 N, anhydrous EtOH, room temperature.

Synthesis of compound 6
To a mixture of 1,10-diaza-18-crown-6 (200 mg, 0.76 mmol) and anhydrous Na2 CO3 (162 mg, 1.52 mmol) in
CH3 CN (1.0 ml) was added a solution of N-(2-bromoethyl)phthalimide (155 mg, 0.61 mmol) in CH3 CN (5.0 ml).
The reaction was conducted under reflux for 36 h. Then the reaction mixture was cooled to room temperature and
filtered. Concentration of the filtrate under reduced pressure and purification by column chromatography (SiO2 ,
CH2 Cl2 /CH3 OH, 30/1–20/1, v/v) gave compound 6 (78 mg, 59%) having 1 H NMR (CD3 OD, 400 MHz) δ
7.88–7.85 (m, 2H), 7.84–7.80 (m, 2H) 3.77 (t, J = 6.8 Hz, 2H), 3.71 (t, J = 5.2 Hz, 4H), 3.62–3.58 (m, 12H),
3.22 (t, J = 5.2 Hz, 4H), 2.84–2.80 (m, 6H); 13 C NMR (CD3 OD, 100 MHz) δ 169.9, 135.4, 133.5, 124.2, 71.4,
71.0, 70.8, 66.8, 55.4, 53.0, 36.9; ESI–MS: m/z 436.78 ([M+H]+ ), 458.73 ([M+Na]+ ) and HR–ESI–MS for
C22 H34 O6 N3 ([M+H]+ ) Calcd: 436.24421; Found: 436.24319.

Synthesis of compound 8
To a mixture of compound 6 (50 mg, 0.11 mmol) and anhydrous K2 CO3 (48 mg, 0.23 mmol) in CH3 CN (3.0 ml)
was added CH3 CH2 I (46 μl, 0.57 mmol). The resulting mixture was refluxed overnight. The reaction mixture was
cooled to room temperature and filtered. Concentration of the filtrate under reduced pressure and purification by
column chromatography (SiO2 , CHCl3 /CH3 OH, 20/1–15/1, v/v) afforded compound 8 (40 mg, 75%) having
1
H NMR (CD3 OD, 400 MHz) δ 7.63–7.58 (m, 2H), 7.46–7.41 (m, 2H), 3.67–3.59 (m, 16H), 3.50 (t, J = 6.4
Hz, 2H), 2.84–2.80 (m, 6H), 2.73–2.69 (m, 6H), 1.04 (t, J = 6.8 Hz, 3H); 13 C NMR (CD3 OD, 100 MHz) δ
176.6, 172.4, 141.1, 135.1, 131.0, 129.2, 129.0, 70.1, 70.0, 68.8, 68.4, 54.7, 53.6, 53.0, 47.6, 38.0, 9.3; ESI–MS:
m/z 464.95 ([M+H]+ ), 486.94 ([M+Na]+ ) and HR–ESI–MS for C24 H38 O6 N3 ([M+H]+ ) Calcd: 464.27551;
Found: 464.27383.

Synthesis of compound 1
To a solution of compound 5 (100 mg, 0.16 mmol) in ethanol (2.0 ml) was added hydrazine hydrate (80%,
100 μl). The resulting solution was heated at 80◦ C for 6 h, and then cooled to room temperature. Concentrated
HCl (0.2 ml) was added and the resulting mixture was refluxed for 30 min. Then the mixture was cooled to room
temperature and filtered. Water (5.0 ml) was added to the residue obtained from the concentration of the filtrate
under reduced pressure. The resulting solution was filtered again. The filtrate was alkalized with saturated LiOH to
pH 10–11, and then extracted with CHCl3 (15 ml × 5). The organic phase was dried over anhydrous Na2 SO4 and
filtered. The filtrate was concentrated under reduced pressure to afford an oily residue (i.e., compound 7) that was
used directly in the subsequent reaction. The removal of the phthalimidyl group was confirmed with ESI–MS: m/z
349.76 ([M+H]+ ). Then, a solution of compound 7 (17 mg, 0.05 mmol), compound 10 (42 mg, 0.12 mmol)
and Et3 N (0.5 ml) in anhydrous EtOH (2.0 ml) was stirred at room temperature for 6 h. The reaction mixture

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 Synthesis & biological evaluation of aza-crown ether–squaramide conjugates as anion/cation symporters Research Article

was centrifuged. The precipitates were collected, washed with ethyl acetate (4 ml × 3) and ethanol (4 ml × 3) and
dried to give compound 1 (18 mg, 39 %) having 1 H NMR (DMSO-d6 , 400 MHz) δ 10.30 (br, 2H), 8.05 (s, 4H),
7.67 (br, 2H), 7.62 (s, 2H), 3.64 (br, 4H), 3.49–3.46 (m, 16H), 2.72–2.68 (m, 12H); 13 C NMR (DMSO-d6 ,
100 MHz) δ 185.2, 180.7, 170.4, 162.7, 141.7, 131.9, 131.6, 124.9, 122.3, 118.4, 115.1, 70.3, 70.0, 55.7, 53.9,
42.7; negative ESI–MS: m/z 961.15 ([M–H]- ) and negative HR–ESI–MS for C40 H41 O8 N6 F12 ([M–H]– ) Calcd:
961.2789; Found: 961.2791.

Synthesis of compound 2
A solution of compound 7 (21 mg, 0.06 mmol), compound 11 (51 mg, 0.18 mmol) and Et3 N (0.5 ml) in
anhydrous EtOH (2.0 ml) was stirred at room temperature for 6 h. The reaction mixture was centrifuged. The
precipitates were collected and washed with ethyl acetate (4 ml × 3) and ethanol (4 ml × 3). The obtained solid
was purified by preparative TLC (CH2 Cl2 /MeOH, 10/1, v/v) to give compound 2 (37 mg, 63%) having 1 H
NMR (DMSO-d6 , 400 MHz) δ 10.88 (br, 2H), 8.46 (br, 2H), 7.71–7.65 (m, 8H), 3.61 (q, J = 5.2 Hz, 4H),
3.49–3.47 (m, 16H), 2.73–2.69 (m, 12H); 13 C NMR (DMSO-d6 , 100 MHz) δ 185.0, 180.3, 170.5, 163.4,
143.4, 127.0, 126.3, 123.6, 122.6, 122.3, 118.1, 70.3, 69.7, 55.8, 54.0, 42.6; ESI–MS: m/z 827.30 ([M+H]+ )
and HR–ESI–MS for C38 H45 O8 N6 F6 ([M+H]+ ) Calcd: 827.3198; Found: 827.3204.

Synthesis of compound 3
To a solution of compound 8 (100 mg, 0.22 mmol) in EtOH (2.0 ml) was added hydrazine hydrate (80%, 66 μl).
The resulting solution was heated at 80◦ C for 6 h, and then cooled to room temperature. Concentrated HCl (0.2 ml)
was added and the resulting mixture was refluxed for 30 min. Then the mixture was cooled to room temperature
and filtered. The residue was obtained from the concentration of the filtrate under reduced pressure and dissolved
in water (5.0 ml). The resulting solution was filtered again. The filtrate was alkalized with saturated LiOH to pH
10–11, and then extracted with CHCl3 (15 ml × 5). The organic phase was dried over anhydrous Na2 SO4 and
filtered. The filtrate was concentrated under reduced pressure to afford an oily yellow residue (i.e., compound 9)
that was used directly in the subsequent reaction. The removal of the phthalimidyl group was confirmed with
ESI–MS: m/z 334.83 ([M+H]+ ). Then, a solution of compound 9 (50 mg, 0.15 mmol), compound 10 (64 mg,
0.18 mmol) and Et3 N (0.5 ml) in anhydrous EtOH (2.0 ml) was stirred at room temperature overnight. The solvent
was removed under reduced pressure and the residue was purified by chromatography on a silica gel column, eluted
with a mixture of CH2 Cl2 and CH3 OH (30/1, v/v) and subsequently by preparative TLC (CH2 Cl2 /MeOH,
10/1, v/v) to give compound 3 (42 mg, 44%) having 1 H NMR (CD3 OD, 400 MHz) δ 8.14 (s, 2H), 7.55 (s, 1H),
3.79 (t, J = 6.0 Hz, 2H), 3.71 (t, J = 5.2 Hz, 4H), 3.64–3.62 (m, 12H), 3.09 (br, 4H), 2.98 (br, 2H), 2.85–2.80
(m, 6H), 1.16 (t, J = 7.2 Hz, 3H); 13 C NMR (CD3 OD, 100 MHz) δ 184.5, 180.7, 170.3, 162.6, 141.2, 132.9,
132.6, 132.2, 131.9, 127.3, 124.6, 121.9, 119.2, 117.8, 114.9, 69.8, 69.6, 68.9, 65.9, 54.8, 53.8, 52.1, 42.2, 8.0;
ESI–MS: m/z 641.61 ([M+H]+ ) and HR–ESI–MS for C28 H39 O6 F6 N4 ([M+H]+ ) Calcd: 641.27683; Found:
641.27417.

Synthesis of compound 4
A solution of compound 9 (46 mg, 0.14 mmol), compound 11 (47 mg, 0.17 mmol) and Et3 N (0.5 ml) in anhydrous
EtOH (2.0 ml) was stirred at room temperature overnight. The solvent was removed under reduced pressure and
the residue was purified by chromatography on a silica gel column, eluted with a mixture of CH2 Cl2 and CH3 OH
(20/1–15/1, v/v) and subsequently by preparative TLC (CH2 Cl2 /MeOH, 10/1, v/v) to give compound 4 (32 mg,
41%) having 1 H NMR (CD3 OD, 400 MHz) δ 7.68 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 3.78 (t, J = 6.0
Hz, 2H), 3.70 (t, J = 5.2 Hz, 4H), 3.64–3.62 (m, 12H), 3.05 (br, 4H), 2.98(br, 2H), 2.84–2.80 (m, 6H), 1.15 (t,
J = 7.2 Hz, 3H); 13 C NMR (CD3 OD, 100 MHz) δ 184.6, 180.5, 170.0, 163.3, 142.3, 128.4, 126.2, 126.2, 125.7,
124.8, 124.4, 124.1, 123.8, 123.0, 120.3, 118.0, 69.6, 69.5, 68.7, 65.9, 54.6, 53.7, 52.0, 42.0, 7.8; ESI–MS: m/z
573.71 ([M+H]+ ) and HR–ESI–MS for C27 H40 O6 F3 N4 ([M+H]+ ) Calcd: 573.28945; Found: 573.28731.

Measurement of association constants & liposomal anionophoric activity

Literature protocols were used to carry out the vesicle preparations [43], 1 H NMR titration [17,37,38,44–50], pH
discharge [51,52], calcein leakage [53], chloride efflux [54,55] and U-tube assay [56] experiments of each of compounds
1–4.

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Research Article Yu, Cai, Hong, Tam, Zhang & Chen

b,c
O O
Na+
Compound 1 + TBACI + NaCIO4 N
O O
N

O N b b N O
H
Cl-
H
d
H H
O N N O
a a
F3C CF3
H H
H c c H
d CF3 F3C d

c
O O
Compound 1 + TBACI N N
O O
O N b
H
b N
H
O d
H Cl- H
O N a a N O
F3C CF3
H H
H c c H

a d CF3 F3 C d b

Compound 1 N
O O
N
c
O O
O N b b N O
H H
H
d
H
O N a N O
a
F3C CF3
H H
H c c H
d CF3 F3 C d
b

12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5

Figure 3. Interaction of compound 1 with chloride and sodium ions. Partial 1 H NMR spectra (CD3 CN, 400 MHz) of (A) compound 1
(1.0 × 10-3 M) in the presence of (B) TBACl (1.1 × 10-3 M) or (C) both NaClO4 (1.0 × 10-3 M) and TBACl (1.1 × 10-3 M).
TBACl: Tetra(n-butyl)ammonium chloride.

Measurement of cytotoxicity & the mechanism of biological actions

Literature protocols were used to carry out the AO staining [22,57–61], MQAE assay [22,29], SBFI AM assay [29],
MTT-based cytotoxicity assay [22], Hoechst staining [22,60,61] and JC-1 staining [22,62].

Results & discussion

Anion recognition
The anion-binding properties of compounds 1–4 were first studied in CD3 CN by means of 1 H NMR titra-
tions [17,37,38]. As shown in Figure 3 and Supplementary Figures 22, 24, 26 and 28, addition of tetra(n-
butyl)ammonium chloride (TBACl) led to significant downfield shifts of the squaramido NHa s and NHb s, and
obvious shifts for the trifluoromethylated aromatic protons. The complexation-induced downfield shifts of the
squaramido NHs and the observation of only one set of downfield-shifted squaramido NHa s and NHb s (Sup-
plementary Figures 23A, 25A, 27A and 29A), strongly suggest that all the squaramido NHs are involved in the
recognition of chloride anions. In the case of compounds 1 and 2, chloride anions may also be bound to either of the
two squaramido subunits, but are under fast exchange between the two squaramido-binding sites. No interaction
between the aza-crown ether moiety and tetra(n-butyl)ammonium (TBA) was observed (Supplementary Figure
30). Molar ratio assays indicated the 1:1 binding modes of compounds 1–4 with chloride anions (Supplementary
Figure 31). This 1:1 binding stoichiometry was further supported by ESI–MS detection (Supplementary Figures
23B, 25B, 27B and 29B). In the negative ESI–MS spectra of compounds 1–4 with TBACl in CH3 CN, in addition
to the ion peaks from the receptor compounds (i.e., m/z 961.78 [1–H]- ), Supplementary Figure 23B), there were
abundant ion peaks that are assignable to the 1:1 complexes with chloride anions (i.e., m/z 997.74 [1+Cl]- ),
Supplementary Figure 23B). These ion peaks with high relative abundances demonstrate the high affinity of com-
pounds 1–4 with chloride anions. It should be noted that no ion peaks from 1:2, 2:1 (receptor:chloride) or any
other higher-order complexes of compounds 1–4 with chloride anions were detected. It has been pointed out that

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Table 1. Calculated lipophilicity (clogP), chloride binding (Ka ) and ion transport efficiency (EC50 , pyranine and
chloride ion selective electrode [ISE] assays) of compounds 1–4.
Compound clogP† Association constants‡ pH discharge§ EC50, 260 s (ISE)¶
Ka (M-1 ) RA1 †† n EC50 (mol%) RA2 †† Na+ K+
1 5.01 (7.13 ± 5.42) × 103 8.2 1.4 ± 0.1 0.14 ± 0.05 24.1 11.6 ± 0.9 7.9 ± 1.1
2 3.25 (2.83 ± 0.21) × 103 3.2 1.3 ± 0.1 1.10 ± 0.16 3.1 17.7 ± 6.2 9.9 ± 1.8
3 2.78 (3.00 ± 0.89) × 10 3
3.4 1.1 ± 0.1 0.77 ± 0.16 4.4 12.1 ± 1.2 7.5 ± 0.4
4 1.91 (8.73 ± 3.05) × 102 1.0 1.3 ± 0.1 3.37 ± 0.82 1.0 30.8 ± 2.4 21.8 ± 3.3
† Calculated using MarvinSketch (Version 6.1.0, Weighted Model, ChemAxon, MA, USA).
‡ Measured by means of 1 H NMR titrations in CD CN. For more details, see Supplementary Figures 22, 24, 26, 28 and 31.
3
§ See Figure 4 and Supplementary Figures 38 and 39 for the detailed measuring conditions.
¶ See Supplementary Figures 41, 42, 46 and 47 and Supplementary Table 2 for the detailed measuring conditions.
†† RA and RA denote the binding affinity and pH discharge efficiency of each compound relative to the least active compound 4, respectively.
1 2

“Job plots should no longer be treated as a golden standard in the analysis of supramolecular systems, and residual
distributions should instead be analyzed to confirm the validity of an assumed model” [63]. Following this notion,
we analyzed the residual distributions to reveal the incorrectness of a 1:2 or 2:1 mode. As shown in Supplementary
Figures 31 and 32, the distributions of sinusoidal residuals are similar in different binding modes. However, the
1:2 and 2:1 fitting produced either high error percentages or negative association constants. These results strongly
suggest that 1:1 binding stoichiometry is more likely than any other binding modes.
The association constants (Ka ’s) of compounds 1–4 with chloride anions were afforded by plotting the chemical
shifts of the squaramido NHs against the concentrations of each compound and using nonlinear least square
fitting according to a 1:1 binding model (Supplementary Figure 31 and Table 1) [63]. These constants demonstrate
that compounds 1–4 show strong affinity toward chloride anions. Notably, the dimeric compounds 1 and 2
exhibit higher affinities than their corresponding monomeric analogs 3 and 4, respectively, suggesting that the
two squaramido subunits bind chloride anions in a cooperative fashion. The ca threefold greater association
constants of compounds 1 and 3 relative to those of compounds 2 and 4, may be rationalized by the stronger
electron-withdrawing ability of 3,5-bis(trifluoromethyl)phenyl substituents than 4-trifluoromethylphenyl groups.
Because sodium perchlorate (NaClO4 ) has high solubility in CH3 CN [64], we evaluated the ion-pairing properties
of compounds 1–4 in the presence of NaClO4 as a source for introducing sodium cations [17,37,38,44–50], by utilizing
1
H NMR titration assays and taking the most active compound 1 as a typical example. As commented by Piatek
et al., “pre-treatment of an ion-pair receptor with a cation salt highly soluble in organic solvents and then titration
of the resulting cation-receptor complex with anion TBA salts, is a standard procedure for studying the effect of
the bound cation on anion complexation for receptors” [65]. Therefore, to clarify whether perchlorate anions can
be bound to compound 1, we first carried out the 1 H NMR titrations of compound 1 with TBA perchlorate
in CD3 CN (Supplementary Figure 33). The negligible changes in the chemical shifts of the aromatic protons
demonstrate that compound 1 does not form a stable complex with perchlorate anions. Then, we studied the
interaction of compound 1 with NaClO4 . As shown in Supplementary Figure 34, addition of NaClO4 to a
solution of compound 1 in CD3 CN led to the upfield shifts of the squaramido NHs, in particular for NHb s.
These upfield shifts were ascribed to the complexation of compound 1 with sodium cations, as compound 1 does
not form a complex with perchlorate anions. Molar ratio assay and ESI–MS detection revealed the 1:1 binding
stoichiometry of compound 1 with sodium cations (Supplementary Figure 35). Addition of TBACl to a solution of
compound 1 and one equivalence of NaClO4 led to significant downfield shifts of the squaramido NHs (Figure 3
& Supplementary Figure 36), suggesting that chloride anions are bound to compound 1. Residual distribution
analysis and nonlinear least square fitting analysis of the relationship between the downfield shifts of the squaramido
NHs and the concentrations of TBACl according to a 1:1 binding model (Supplementary Figure 36), afforded
the association constant of compound 1 with chloride anions being 2.18 × 103 M-1 in the presence of one
equivalence of NaClO4 , over threefold smaller than that in the absence of NaClO4 (Table 1). This reduced binding
affinity may be ascribed to the formation of a contact ion pair that weakens the interaction of compound 1 with
chloride anions [38,50], and strongly suggests that sodium cations are involved in the recognition of compound
1 toward chloride anions. It should be noted that in the presence of TBACl, NaClO4 had different effects on
the squaramido NHa s and NHb s of compound 1. Under the conditions depicted in Supplementary Figures 22
and 36, the addition of NaClO4 led to the downfield shift of the squaramido NHa s from δ = 2.756 p.p.m. to

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Research Article Yu, Cai, Hong, Tam, Zhang & Chen

1.0 0.5
Compound 1
Compound 2 Compound 1
Compound 3 Compound 2
0.8 0.4 Compound 3

Relative chloride efflux

Compound 4
Control Compound 4
Triton
Control
X-100
Relative FI

0.6 0.3

0.4 0.2

0.2 0.1

0.0
0.0
0 5 10 15 20 0 50 100 150 200 250 300
T (min) T (s)

Figure 4. The liposomal ion transport activity of compounds 1-4. (A) Plots of the relative fluorescence intensity of egg-yolk
L-α-phosphatidylcholine liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt against time in the presence of
compounds 1–4 (0.25 mol%). Intravesicular conditions: 0.1 mM 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt and 50 mM NaCl in
25 mM HEPES buffer (pH 7.0); extravesicular conditions: 50 mM NaCl in 25 mM HEPES buffer (pH 8.0). Ex 460 nm; em 510 nm. (B) Plots of
the relative chloride efflux against time in the presence of compounds 1–4 (5 mol%) in egg-yolk L-α-phosphatidylcholine vesicles loaded
with 500 mM NaCl in 25 mM HEPES buffer (pH 7.0) and dispersed in 500 mM NaNO3 in 25 mM HEPES buffer (pH 7.0).

δ = 2.785 p.p.m. (δ = 0.029 p.p.m.), and the squaramido NHb s from δ = 1.683 p.p.m. to δ = 1.919 p.p.m.
(δ = 0.236 p.p.m.). This result suggests that sodium cations are complexed to the aza-crown ether moiety and
such complexation has more profound effects on the adjacent squaramido NHb s than the distant NHa s.
Taken together, the above results suggest that compound 1 is able to form an ion-pair complex with chloride
anions and sodium cations. The proposed interaction mode of compound 1 with cations and anions is shown in
Figure 1.

Liposomal anion/cation transport

Ionophoric activity
The ion transport properties of compounds 1–4 on liposomal models was first studied by using a conventional pH
discharge assay based on HPTS (pKa 7.2) [51,52]. It can be seen from Figure 4 that addition of each of compounds 1–4
to EYPC-derived liposomes (100 nm diameter, extrusion) containing an internal pH of 7.0 and an external aqueous
phase of pH 8.0, led to an increase in the fluorescence intensity of HPTS. This observation indicates that compounds
1–4 are able to discharge the pH gradients across the liposomal membranes and increase the intravesicular pH.
Plots of the initial rate constants (kin ’s) against the concentrations of each compound gave the Hill’s coefficient n
and the EC50 value (Table 1, Supplementary Figures 38 & 39 & Supplementary Table 1). The Hill’s coefficient
n being around 1 suggests that compounds 1–4 exert their ionophoric activity in a unimolecular fashion. The
EC50 values indicate that these conjugates exhibit effective pH discharge activity. Calcein leakage experiments
suggest that compounds 1–4 do not cause the leakage of this dye out of the EYPC vesicles (Supplementary Figure
40), indicating that the observed pH discharge activity is induced by the compounds [53]. Of the four conjugates,
3,5-bis(trifluoromethyl)phenyl compounds 1 and 3 are eight- and fourfold more active than the corresponding
4-trifluoromethylphenyl analogs 2 and 4, respectively. Dimeric conjugates 1 and 2 exhibit six- and threefold higher
transport efficiency than the corresponding monomeric conjugates 3 and 4, respectively.
It is well recognized that anion-binding ability and lipophilicity are two major factors that may regulate the
anionophoric activity of an anion transporter [26,51,66–68]. The pH discharge activity of compounds 1–4 correlates
well with their binding affinity toward chloride anions, suggesting that anion-binding affinity is one of the factors
that regulate the anion transport. Then, we calculated the partition coefficients (clogP) of compounds 1–4 (Table 1).
Analysis of the correlation between the clogP and the EC50 values suggests that lipophilicity has limited impact on

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 Synthesis & biological evaluation of aza-crown ether–squaramide conjugates as anion/cation symporters Research Article

50

40
ol %)

30
EC 50 (m

20

10

R
b
0

+
4

K
+
3

Co

n
tio
mp

N
a
+
ou

Ca
2

nd

Li
+
1

Figure 5. Effect of alkali metal cations on the chloride efflux efficiency (EC50 , 260 s) of compounds 1–4 in egg-yolk
L-α-phosphatidylcholine vesicles containing an intravesicular 500 mM MCl solution (25 mM HEPES, pH 7.0) and
extravesicular 500 mM MNO3 solution (25 mM HEPES, pH 7.0).
M: Li, Na, K or Rb.

the pH discharge activity of compounds 2–4, but may regulate the anion transport efficiency of compound 1. This
may account for why the most lipophilic compound 1 exhibits the highest anion transport efficiency.

Anion-selective transport activity

To address whether compounds 1–4 are able to transport anions, we monitored the efflux of chloride anions out of
the EYPC liposomes by using a chloride ion selective electrode [54,55]. As shown in Figure 4B and Supplementary
Figures 41 and 42, addition of a sample of each of compounds 1–4 (of varying concentrations to lipid) in DMSO
to the EYPC vesicles loaded with NaCl, led to the concentration-dependent chloride efflux. This result suggests
that compounds 1–4 are capable of mediating the transmembrane transport of chloride anions.

Mechanism of anion/cation symport

To gain insights into the probable mechanism of action of compounds 1–4, we replaced the external nitrate anions
with sulfate anions in the above chloride efflux experiments, and re-measured the chloride efflux by means of
chloride ion selective electrode technique (Supplementary Figure 43) [18]. Sulfate anions have high dehydration
energy (G = -1080 kJ/mol-1 ), and therefore, are not readily transported through a lipid bilayer [18,69]. As a
result, no inhibitory effect of sulfate anions on the chloride efflux was observed. In addition, no effect of HCO3 -
was observed, either. No essential dependence of the chloride efflux rate on the nature of the external anions was
observed, suggesting that anion exchange is not a predominant step in the permeation process. Compounds 1–4
behaving as anion/cation symporters were further supported by the chloride efflux in the presence of different
alkali metal ions (Li+ , Na+ , K+ or Rb+ ; Supplementary Figures 41, 44, 46 & 48) [18]. Analysis of the relative
chloride efflux at 260 s according to a Hill’s equation gave the chloride efflux efficiency (EC50, 260 s ) of compounds
1–4 (Figure 5, Table 1, Supplementary Figures 42, 45, 47 & 49 & Supplementary Table 2). As a consequence, the
chloride efflux efficiency varies with the alkali metal cations with certain level of selectivity for potassium cations
in liposomal models. These results strongly suggest that compounds 1–4 facilitate the transport of anions via an
anion/cation symport process.
In addition, to clarify whether compounds 1–4 function as mobile carriers or channels, we first used the widely
used cholesterol assays to probe the probable mode of action by measuring the pH discharge across the vesicular
membranes formed from EYPC and cholesterol (7/3 molar ratio) [56]. As shown in Supplementary Figure 50, a
dramatic reduction in the pH discharge activity was observed. This is a typical behavior that is generally displayed

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Research Article Yu, Cai, Hong, Tam, Zhang & Chen

by a mobile carrier. Because the results derived from cholesterol assays are sometimes inconclusive [56], we further
used a U-tube assay to re-confirm the probable mechanism of mobile carriers (Supplementary Figure 51). To
accomplish this assay, aqueous sodium chloride solution and sodium nitrate solution were used as the resource
phase and the receiving phase, respectively, and they were separated by a bulk nitrobenzene phase. The changes
in the concentration of chloride anions in the receiving phase were monitored by using a chloride ion selective
electrode and are evidence for whether chloride anions can be transported through the nitrobenzene phase. As a
consequence, higher concentration of chloride anions was observed with compounds 1–4 than without them. This
transport of chloride anion through the bulk organic phase, the decreased pH discharge indicated by the cholesterol
assays and the Hill’s coefficients n being around 1 in the liposomal model experiments (Table 1 and Supplementary
Figure 39), were taken together to demonstrate that compounds 1–4 mediate the transmembrane anion/cation
symport most probably via a mobile carrier mechanism.

In vitro anion/cation transport

The above pH discharge experiments indicate that compounds 1–4 are able to promote the deacidification of the
EYPC vesicles. This result inspired us to investigate their ability to deacidify acidic organelles, such as lysosomes at
cellular levels. For this purpose, we used AO to stain human cervical cancer HeLa cells and observed the changes in
the fluorescence of AO. Because AO exhibits characteristic pH-sensitive fluorescence that changes from orange to
green when pH increases, it has been widely used as a fluorescent reporter for the pH change within organelles [57–59].
As shown in Supplementary Figure 52, vital staining of the HeLa cells with AO led to the presence of granular
orange fluorescence in the cytoplasm of organelles. After the cells were treated with compounds 1–4, we observed
the complete disappearance of the orange fluorescence for compounds 1 and 3, and a significant reduction in the
orange fluorescence for compounds 2 and 4. This result suggests that compounds 1 and 3 have higher ability
to alkalize lysosomes than compounds 2 and 4, which parallels their anionophoric activity observed in liposomal
models (Table 1).
To gain insight into whether the lysosomal deacidification induced by compounds 1–4 was due to the influx
of anions and/or cations into cells, we first used an MQAE-based fluorescent assay to monitor the entry of
chloride anions into HeLa cells facilitated by the two active compounds 1 and 3 [21,62,70]. As a cell-permeable dye,
MQAE exhibits chloride-selective fluorescence, and thus, the fluorescence quenching in the HeLa cells after treated
with compound 1 or 3, may provide direct evidence for the influx of chloride anions into the HeLa cells. As a
consequence, significant quenching of the MQAE fluorescence was observed after the HeLa cells were incubated
with compound 1 or 3 (Figure 6A). These data demonstrate that the influx of chloride anions into the intracellular
matrix may be mediated by these two compounds.
It is known that extracellular sodium concentration is much higher than intracellular sodium concentration (145
vs 12 mM) [71]. Similarly, extracellular chloride concentration is higher than intracellular chloride concentration
(120 vs 4–60 mM) [71]. These concentration differences make it possible for compounds 1 and 3 to facilitate the
entry of sodium cations and chloride anions into cells. To test this hypothesis, we used a sodium-selective fluorescent
indicator SBFI-AM to confirm that compounds 1 and 3 are able to mediate the entry of sodium cations into cells
(Figure 6B) [21,62]. Incubation of the HeLa cells with SBFI-AM, followed by the treatment with compound 1 or 3 led
to a dramatic increase in the fluorescence. This result indicates that compounds 1 and 3 can efficiently increase the
intracellular sodium concentration. It is noteworthy that whether the influx of chloride anions and sodium cations
into the HeLa cells was mediated by compounds 1 and 3 themselves or by their biological channels/transporters
activated by compound 1 or 3 remains to be clarified.

Biological activity
Cytotoxicity
It has been reported that the coupled entry of anions and cations into cells is able to trigger apoptosis [21].
Therefore, we used a conventional MTT assay to measure the cytotoxicity of compounds 1–4 toward four human
solid tumour cells (i.e., HeLa, lung adenocarcinoma epithelial A549, human breast cancer MCF-7 and human liver
cancer HepG2 cells) and LO2 human normal liver cells. The inhibitory activity of each compound at 50 μM was
shown in Supplementary Table 3 and Supplementary Figure 53, and the IC50 values that were calculated from the
dose-dependent profiles (Supplementary Figures 54–56) are listed in Table 2 and Supplementary Tables 3–6. Here
the IC50 value is defined as the concentration of each compound that produces 50% inhibition in cell growth.
Doxorubicin was used as a positive control.

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100

Normalized FI of MQAE
80

60

40

20

0
Control 1 3

Figure 6. Changes in the intracellular concentrations of 200

chloride anions and sodium cations. Normalized FI of (A)
MQAE and (B) SBFI-AM in HeLa cells incubated with Normalized FI of SBFI AM 160
MQAE (5 mM) or SBFI-AM (10 μM) for 3.5 h followed by
the treatment with 10 μM of compound 1 or 3 for 2 h.
120
Fluorescence intensity was recorded with the plate
reader at λem = 460 nm (λex = 350 nm) for MQAE or
λem = 500 nm (λex = 340 nm) for SBFI-AM and reported 80
relative to the fluorescence intensity of untreated cells.
The mean intensity of three independent experiments 40
was taken for each data point represents.
FI: Fluorescence intensity; MQAE:
N-(ethoxycarbonylmethyl)-6-methoxy quinolinium 0
bromide; SBFI-AM: Sodium-binding benzofuran Control 1 3
isophthalate acetoxymethyl ester.

Table 2. Cytotoxicity of compounds 1–4 and doxorubicin.

Compound Cytotoxicity (IC50 , μ M)†
HeLa A549 MCF-7 HepG2 LO2
1 26.7 ± 1.2 ⬎50 34.2 ± 3.4 ⬎50 ⬎50
2 ⬎50 ⬎50 ⬎50 ⬎50 ⬎50
3 19.1 ± 0.3 30.2 ± 0.4 31.4 ± 3.9 14.6 ± 0.3 21.8 ± 1.9
4 ⬎50 ⬎50 ⬎50 ⬎50 ⬎50
Doxorubicin 0.13 ± 0.01 0.51 ± 0.06 0.14 ± 0.01 1.9 ± 0.4 0.17 ± 0.03
† TheIC50 values of ⬎50 μ M were estimated because of the low inhibitory activity in the initial single-dose screening (50 μ M). See Supplementary Figure 53 and Supplementary Tables
4–6 for more details.

It can be seen that compounds 1 and 3 exhibit promising cytotoxicity, in particular towards HeLa and MCF-7
cells, whereas compounds 2 and 4 show lower cytotoxic effects. The cytotoxicity of compounds 1–4 roughly
correlates with their anionophoric activity observed both in liposomal models and cells, which implies that the
anion/cation symport may play a crucial role in the cytotoxic effect. It should be noted that though compound
1 is a stronger transporter than compound 3, it is less cytotoxic. This result implies that the cytotoxic effect of
compound 3 is not purely due to anion/cation symport [72]. In addition, it appears that compound 1 exhibits some
level of selectivity for HeLa and MCF-7 cancer cells over the normal LO2 cells. More future work will be done to
confirm this.

Effect of chloride anions & sodium cations on cytotoxicity

It is known that dysregulation of ionic homeostasis in live cells may lead to cell death [21,62,70]. As shown above,
compounds 1–4 are able to deacidify acidic organelles and facilitate the entry of both chloride anions and sodium
cations into live cells. Therefore, to evaluate the cytotoxic effects of these in vitro ion transport processes on the live

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Research Article Yu, Cai, Hong, Tam, Zhang & Chen

cells, we measured the cytotoxicity of the two active compounds 1 and 3 toward HeLa cells in the presence and the
absence of chloride anions or sodium cations. As shown in Supplementary Figure 57, both compounds 1 and 3
are more cytotoxic toward the HeLa cells in the presence of chloride anions or sodium cations than in the absence
of chloride anions or sodium cations. This enhanced cytotoxicity, together with the facilitated entry of chloride
anions and sodium cations into cells by compounds 1 and 3, strongly suggests that chloride/sodium symport plays
a significant role in the cytotoxic effects [21].

Hoechst 33342 staining

Literature reports indicate that cell death may be primarily divided into necrosis, apoptosis and autophagy [73]. Thus,
to gain insights into the probable mechanism of biological action, we first conducted Hoechst 33342 staining of
HeLa cells treated with compounds 1 and 3 (Supplementary Figure 58). Hoechst 33342 is a specific DNA
fluorescent probe that can be used to identify changes in the nuclear morphology by fluorescence microscopy [60,61].
Compared with the untreated cells, the HeLa cells treated with compounds 1 and 3 displayed stronger blue
fluorescence accompanied with the change of nuclear morphology, including nuclear condensation, fragmentation
and formation of apoptotic bodies. In addition, ‘bean-shaped’ nuclei were also observed. These characteristics are
hallmarks that compounds 1 and 3 induce cell death probably via an apoptotic pathway.

JC-1 staining
The probable apoptosis mechanism of action of compounds 1 and 3 was further confirmed by JC-1 staining of HeLa
cells. JC-1 is a cell-permeable, fluorescent dye that is very sensitive to the change in the mitochondrial membrane
potential and a loss or decrease of the mitochondrial membrane potential is a hallmark of apoptosis [62,74]. As shown
in Supplementary Figures 59 and 60, treatment of the HeLa cells with compound 1 or 3 of varying concentrations
and then with JC-1, led to a significant reduction in the red-to-green fluorescent intensity ratio of JC-1. This
reduction indicates that the mitochondrial membrane potential decreased when the HeLa cells were treated with
compounds 1 and 3, and provides evidences for apoptotic cell death at an early stage.

Conclusion
In summary, we have successfully synthesized four aza-crown ether–squaramide conjugates and confirmed their
structures on the basis of NMR (1 H and 13 C) and ESI–MS (LR and HR) data. We have studied their anion
recognition by means of 1 H NMR titrations and MS and found that they are able to form stable ion-pair
complexes. We have investigated their anionophoric activity and ion selectivity on liposomal models by using
fluorescence assays and chloride ion selective electrode techniques. The results have indicated that these conjugates
are able to facilitate the transmembrane transport of anions via an anion/cation symport process. We have measured
the cytotoxicity of these compounds towards four cancer cells and one type of normal cells by means of MTT
assays. As a result, these conjugates exhibit moderate cytotoxicity toward the tested solid tumor cell cells. Of the four
conjugates, the two compounds with 3,5-bis(trifluoromethyl)phenyl substituents exhibit higher anion transport
and cytotoxicity than their corresponding analogs bearing 4-trifluoromethylphenyl substituents. Mechanistic study
has shown that these conjugates are able to alkalinize the acidic organelles, disrupt the cellular homeostasis of
chloride anions and sodium cations and induce cell death most probably via an apoptotic mechanism. Thus,
these conjugates are exploitable as apoptotic agents that exert their bioactivity through a mechanism of action of
anion/cation symport across cellular membranes.

Future perspective
Sodium cations and chloride anions are the most abundant cations and anions in natural systems, respectively,
and play a crucial role in maintaining normal physiological function. The design and synthesis of anion/cation
symporters are expected to help to enhance our understanding of how anion/cation symporters function in
biological systems and may provide some useful guidance for the rational design of new antitumor drugs that
function via the transmembrane transport of anions and cations.

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.future-science/doi/suppl/10.
4155/fmc-2018-0595

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 Synthesis & biological evaluation of aza-crown ether–squaramide conjugates as anion/cation symporters Research Article

Acknowledgements
The authors give their thanks to all those who have helped with this issue. Listed are authors, referees and others who have kindly
given their time, effort and expertise; their generosity has helped establish this publication: XH Yu, XJ Cai, XQ Hong, KY Tam, K
Zhang, WH Chen.

Financial & competing interests disclosure

This work was financially supported by the National Natural Science Foundation of China (No. 21877057). The authors have no
other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined
in the Declaration of Helsinki for all human or animal experimental investigations.

Summary points
• Four aza-crown ether–squaramide conjugates having 3,5-bis(trifluoromethyl)phenyl or 4-trifluoromethylphenyl
substituents were designed and synthesized as anion/cation symporters.
• These compounds are able to bind chloride anions and form stable ion-pair complexes.
• These compounds are capable of promoting the transport of anions across lipid bilayer membranes via an
anion/cation symport process and as mobile carriers.
• These compounds are able to disrupt the cellular homeostasis of chloride anions and sodium cations and induce
the basification of acidic organelles in live cells.
• These conjugates exhibit moderate cytotoxicity toward the tested human cancer cells. Of them, the
3,5-bis(trifluoromethyl)phenyl conjugates are more active as both anion transporters and cytotoxic agents than
the corresponding 4-trifluoromethylphenyl analogs.
• These compounds are capable of mediating the influx of chloride anions and sodium cations into live cells to
trigger cell death most probably via an apoptotic pathway.

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