Organic Letters
Letter
based on the concentration profiles of 1a−1c and prodigiosin,
provided effective concentration at 50% activity (EC ) =
can be proposed as the preferred HCl binding site of the
receptor as the site has considerable advantage due to
5
0
1
8
19
0
3
6.64, 14.90, and 79.48 pM), respectively (Table S2)
cooperative and microenvironmental effects, and the
receptor can also provide significant hydrophobic covering to
+
−
suggesting that the alkyl groups play an important role in
increasing the cLogP values of the molecules, thereby
improving the membrane permeability. Interestingly, the
calculated transporter to lipid ratios at the EC50 values of 1b
and 1c were better than that of prodigiosin. Considering these
results, the activities of 1b and 1c can be considered better
than all reported synthetic HCl transporters because the
highest active synthetic HCl transporter hitherto reported in
polar H and Cl . On the basis of these insights, geometry
+
−
optimization for the [1c + H + Cl ] was performed by
Gaussian 09 program using B3LYP functional and 6-31G(d,p)
2
6
+
−
basis set. The optimized structure of the [1c + H + Cl ]
complex indicated that the protonated form of 1c (protonated
−
−
−
−
1
1
11
3
12
+
−
The excellent H /Cl cotransport activity of 1c inspired us
to evaluate its effect on biological systems. 1a−1c and
prodigiosin were incubated in epithelial human breast cancer
cells MCF-7 and noncancerous HEK293T cells for 24 h, and
2-yl)-2,5-diphenyl tetrazolium bromide) assay (Figure S44).
The results showed that 1c and prodigiosin are highly toxic to
the MCF-7 cells (IC50 = 1.0 μM for 1c and prodigiosin, Figure
2
3
literature trails the activity of prodigiosin by a small margin.
The calculated Hill coefficient n values of 1.17, 0.99, and 1.00
for 1a−1c indicate that one molecule is sufficient to form the
active transporter.
Further studies were carried out with the most efficient
transporter 1c. During the transport studies using 1c (c = 15
+
pM) across EYPC−LUVs ⊃ HPTS, a variation of cations (Li ,
+
+
+
+
22
Na , K , Rb , and Cs ) in the extravesicular buffer, provided
−
−
−
−
−
contrast, the variation of anions (F , Cl , Br , I , OAc , and
−
22
3
−
To investigate the operative transport mechanism, valino-
mycin and monensin coupled experiments were performed
2
4
across EYPC−LUVs using a chloride ion-selective electrode.
−
We found that Cl efflux is only observed in the presence of
monensin but not valinomycin (Figure 3B,C). This result
+
+
signifies that K /H antiport by monensin cooperatively
+
−
couples to H /Cl cotransport promoted by 1c, leading to
overall KCl efflux from the liposomes. Particularly, the lack o+f
chloride efflux in the presence of valinomycin explains that H
−
and Cl transport by 1c is an inseparable process. It suggests
that 1c behaves like prodigiosin where chloride cannot be
transported without a proton also being transported, and
protonated 1c cannot diffuse through the lipid bilayer without
Figure 4. Cellular viability (assessed by the MTT assays) for 1c and
prodigiosin in (A) MCF-7 and (B) HEK293T cells. **, p < 0.001, and
2
4c
+
−
a counteranion.
The H /Cl symport nature of 1c was
*
**, p < 0.0001, when compared to untreated cells. Viability of MCF-
further verified by investigating the effect of valinomycin on
−
7 cells was assessed in the presence and absence of chloride anions in
extracellular media (HBSS buffer) for 1c (C). MCF-7 cells stained
with acridine orange after incubation at 0 μM (D) and 2.0 μM (E)
compound 1c for 24 h.
the Cl influx activity of 1c across EYPC−LUVs ⊃ lucigenin,
+
22
also performed U-tube experiments to confirm that 1c
1
9d
lesser in noncancerous HEK293T cells when compared to that
Experimental determination of pK values of 1c was not
of prodigiosin at concentrations >1.0 μM (Figure 4B). To gain
a
−
successful possibly due to its sparing water solubility and very
further insight in support of Cl -ion-mediated cell death, we
close pK values of N and N centers, as estimated by the
measured the cytotoxicity of 1c toward MCF-7 cells in the
a
11
8
−
program Schrodinger (i.e., pK = 4.92 and 4.72 for N and N
presence and absence of Cl ions (Figure 4C). Remarkably
a
11
8
2
5
−
respectively). Therefore, to understand the HCl binding
mode, involved in membrane transport, the activity of 1c was
compared with 1d. A bare minimum activity of 1d indicates
the involvement of N11 in the transport process (Figure 3A).
Furthermore, the cocrystal of 1a·HCl demonstrates the mode
of a proton−chloride recognition motif involving the
protonation of the pyridine N-center (Figure 3D). The cavity
formed by benzimidazole and pyridine moieties accommodates
higher cytotoxicity in the presence of Cl ions in the
−
extracellular media strongly suggests that Cl -ion-mediated
cell death is one of the major factors responsible for the
−
cytotoxic effects. It is known in the literature that such Cl -ion-
mediated cell death triggers the apoptotic pathways in live
22
+
−
cells. Since 1c displayed the H /Cl cotransport across the
bilayers, we anticipated that it might have a direct impact on
the lysosomal pH. To investigate, acridine orange (AO, a cell-
permeable pH-sensitive dye) was used to monitor the
lysosomal pH changes in cells treated with 1c. AO shows a
characteristic orange emission upon accumulation in acidic
compartments like lysosomes but shows a green fluorescence
−
a Cl through hydrogen-bonding interactions, which was
−
−
evident from the distances N ···Cl = 3.100 Å and N ···Cl =
3
11
3
.074 Å featuring a perfect consequence of positive
cooperativity. The pocket involving the N and N centers
3
11
6
133
Org. Lett. 2021, 23, 6131−6136
Mondal, Abhishek
