Tripodal squaramide conjugates as highly effective transmembrane anion transporters

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx

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Tripodal squaramide conjugates as highly effective transmembrane anion transporters Xiong-Jie Cai, Zhi Li, Wen-Hua Chen ⇑ Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China

a r t i c l e

i n f o

Article history: Received 11 January 2017 Revised 18 February 2017 Accepted 6 March 2017 Available online xxxx Keywords: Anionophore Squaramide Anionophoric activity Ion selectivity

a b s t r a c t Two tripodal squaramide conjugates having 4-(trifluoromethyl)phenyl and 3,5-bis(trifluoromethyl)phenyl substituents were synthesized and found to exhibit highly efficient transmembrane anion transport with the EC50 values being 0.14 and 0.75 mol%, respectively. Though one of them has been reported to act as a strong anion receptor, in particular for sulfate anions, these two compounds exhibit no significant selectivity with respect to the tested monovalent anions and a very low level of activity in the presence of sulfate anions. Ó 2017 Elsevier Ltd. All rights reserved.

Anions are ubiquitous in nature and transport of anions across cell membranes plays a crucial role in the natural functioning of biological systems.1 This biological importance has triggered wide interest from medicinal chemists to create synthetic transmembrane anion transporters.2 Such small-molecule compounds may serve as alternatives for defective natural anion channels, by replacing the missing anionophoric activity.3 In addition, some synthetic anion transporters have been found to have high potentials as drug candidates for the treatment of cancers4 and bacterial infections,5 in which the normal anion balance in cancer or bacteria cells is destroyed by an effective anion transporter. Several strategies, such as configuration,6 flexibility7 and lipophilicity,8,9 have been successfully employed to construct effective anion transporters. Remarkable among them is tripodal conjugation as a useful approach to create effective anion transporters.10 Such conjugation is characteristic of a well-dispersed but convergent 3D array of hydrogen-bonding donor functionality, leading to an increase in the number of anion binding sites and thereby potent anion receptors, in particular for oxo-anions such as sulfate anions.11 For example, Davis et al. have shown that tris (2-aminoethyl)amine-based receptors with appended catechol groups, are capable of transmembrane chloride transport.10a Gale et al. have reported that tris(2-aminoethyl)aminebased tripodal tris-thiourea receptors are capable of chloride/

⇑ Corresponding author. E-mail address: [email protected] (W.-H. Chen).

bicarbonate transport and as such represent a new class of bicarbonate transport agents.10b Recently, Jin et al. have reported the synthesis of squaramidebased tripodal anion receptors, like compound 1 having 4-(trifluoromethyl)phenyl substituents (Fig. 1).12 These tripodal conjugates possess enriched anion binding sites and have been found to strongly and selectively encapsulate SO24 over the other examined anions via hydrogen-bonding interactions. The results imply that these receptors may find potential applications in various fields, such as anion transporters and extraction agents. However, whether they function as transmembrane anion transporters remains to be established.13 In addition, it is also of fundamental interest to address whether such powerful receptors for a given anion would serve as potent transmembrane transporters for that anion. Specifically, we are concerned about whether these tripodal squaramide conjugates exhibit potent anion transport activity and in particular selective transport of sulfate anions over other anions.14 With these thoughts in mind, herein we report the synthesis and anion transport properties of this compound 1 and the analog 2 having 3,5-bis(trifluoromethyl)phenyl groups. Compounds 1 and 2 were synthesized according to the approach depicted in Scheme 1. Thus, reaction of 3,4-diethoxycyclobut-3-ene-1,2-dione with 4-(trifluoromethyl)aniline and 3,5-bis(trifluoromethyl)aniline in EtOH, in the presence of zinc trifluoromethanesulfonate (Zn(CF3SO3)2),13a afforded compounds 3 and 4 in 57% and 63% yields, respectively. Reaction of compounds 3 and 4 with tris(2-aminoethyl)amine in the presence of Et3N, gave compounds 1 and 2 in 81% and 41% yields, respectively. Compound 1 shows structural data that are

http://dx.doi.org/10.1016/j.bmcl.2017.03.024 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

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X.-J. Cai et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx

O

NHR

O

N H

To quantitatively characterize the ion-transporting efficiency of compounds 1 and 2, we carried out concentration-dependent pH discharge experiments.16 In this experiment, a pH sensitive dye, pyranine was encapsulated within large unilamellar EYPC vesicles and used as a fluorescence-responsive reporter of the pH changes within the vesicle interior, due to the transmembrane transport of proton or hydroxide.17 As shown in Figs. 2b and S8, addition of compounds 1 and 2 of varying concentrations to the EYPC liposomal dispersions containing an internal pH of 7.0 and an external aqueous phase of pH of 8.0, led to a rapid increase in the pyranine fluorescence, indicating that these two compounds were capable of inducing pH discharge across the membrane. The initial rate constants (kin’s) of compounds 1 and 2 at each concentration were calculated from the concentration-dependent profiles. Nonlinear curve fitting of the initial rate constants against the concentrations of compounds 1 and 2 according to a Hill equation, kin = k0 + kmax[compound]n/([compound]n + [EC50]n), gave the Hill coefficient n and EC50 values of each compound (Table 1). Here EC50 is defined as effective transporter loading that needs to reach 50% of the maximum rate (kmax) after a specified time period and thus a measure of the effectiveness of a given transporter.18 The EC50 values of compounds 1 and 2 being 0.14 mol% and 0.75 mol%, respectively, indicate that both conjugates are very active and represent an effective class of anion transporters. Though less active than some tripodal urea and thiourea-based conjugates having EC50 values as low as 0.0044 mol%,10b compounds 1 and 2 are comparable to C3-symmetric benzoxazine urea (EC50 = 0.49 mol%),19 and much more active than cyclopeptide-based tris-thioureas (EC50 = 1.13  3.11 mol%),14 phosphoric triamide-based receptors (EC50 = 6.2  7.0 mol%),10a our squaramido-functionalized bis(choloyl) conjugates (EC50 = 2.6  2.8 mol%)13a and squaramide-linked bis(choloyl) conjugate (EC50 = 3.82 mol%).13e The n values of 1.27 for compound 1 and 1.17 for compound 2, respectively, indicate that these two compounds do not aggregate to function.

O N

O

HN

O

CF3

NHR

NH

RHN

1: R =

CF3

2: R = CF3

O

Fig. 1. Structures of tripodal squaramide conjugates 1 and 2.

in agreement with the ones reported in literature,12 whereas compound 2 was fully characterized on the basis of ESI MS (LR and HR) and NMR (1H and 13C) data (See Supporting Information). The anion-transporting activity of compounds 1 and 2 was firstly examined by directly measuring the chloride efflux across liposomal membranes derived from egg-yolk L-a-phosphatidyl choline (EYPC), by means of chloride ion selective electrode technique.15 In this experiment, a series of large unilamellar EYPC vesicles (100 nm diameter, extrusion) loaded with sodium chloride, were prepared and suspended in an external isotonic NaNO3 solution. After a sample of compound 1 or 2 (of varying concentrations in mol% relative to the EYPC concentrations) was added as a DMSO solution, the efflux of chloride was monitored by a chloride ion selective electrode. If compound 1 or 2 functions as a chloride transporter, chloride will be released from the vesicles and can be detected by the electrode. After 300 s, the vesicles were lysed by addition of 5 wt% Triton X-100 and the final reading of the electrode was used to calibrate 100% release of chloride. The data are shown in Figs. 2a and S7, which indicate that compounds 1 and 2 are capable of releasing chloride under the measuring conditions and that the rate of chloride efflux increases in a concentration dependent fashion.

O O

O O

(i)

NHR

O O

O

NHR

O

N H

O (ii)

O

3, 4

N NH

RHN O

HN

O NHR

1, 2

1, 3: R =

CF3 CF3

2, 4: R = CF3

O

Scheme 1. Synthesis of compounds 1 and 2. Reagents and conditions: (i) substituted phenylamine, Zn(CF3SO3)2, anhydrous ethanol, room temperature, 10 h; (ii) tris(2aminoethyl)amine, Et3N, anhydrous ethanol, room temperature, 6 h.

Fig. 2. (a) Relative chloride efflux promoted by compound 1 of varying concentrations under the measuring conditions of 25 mM HEPES (pH 7.0, 500 mM NaCl) for internal vesicles and 25 mM HEPES (pH 7.0, 500 mM NaNO3) for external vesicles. The experiment that was conducted in NaNO3 media and in the absence of compound 1, was used as a control. (b) Discharge of a pH gradient across EYPC-based liposomal membranes in the presence of compound 1 of varying concentrations. Intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (50 mM NaCl, pH 7.0); Extravesicular conditions: 25 mM HEPES (50 mM NaCl, pH 8.0). Ex 460 nm; em 510 nm. These and all the after-mentioned experiments were performed in triplicate, and the mean values were taken.

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X.-J. Cai et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx Table 1 Kinetic parameters for the pH discharge by compounds 1 and 2 and their binding affinity (Ka, M Compound

n

1

) toward tetrabutylammonium salts.

EC50 (mol%)a Cl

log Ka Br

I

NO3

1

1.27 ± 0.32

0.14 ± 0.06

0.13 ± 0.07

0.06 ± 0.03

0.11 ± 0.04

2

1.17 ± 0.18

0.75 ± 0.29

0.49 ± 0.30

0.05 ± 0.03

0.23 ± 0.04

SO24

Cl b

2.84 ± 0.06 2.65 ± 0.09c 3.47 ± 0.05b

5.64 ± 0.26b 4.87 ± 0.07c >5.0d

a

Measured in EYPC vesicles under the measuring conditions of internal vesicles: 0.1 mM pyranine in 25 mM HEPES (50 mM NaX, pH 7.0) and external vesicles: 25 mM HEPES (50 mM NaX, pH 8.0) (Fig. S8-11). Here X = Cl, Br, I and NO3. b Measured by means of spectrophotometric titrations in DMSO (Fig. S15a-c and Table S5). c The data were taken from Ref. 12 and measured by means of 1H NMR titrations in DMSO. d The spectrophotometric titration profiles of compound 2 with sulfate anions suggest that it shows apparent binding affinity that exceeds the detection limit (105 M 1) of spectrophotometric titration (Fig. S15d).

Fig. 3. (a) Relative fluorescence intensity (FI) of pyranine in the presence of compound 1 of varying concentrations. Intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (pH 7.0, 50 mM Na2SO4) and extravesicular conditions: 25 mM HEPES (pH 8.0, 50 mM Na2SO4). (b) Relative chloride efflux promoted by compound 1 (1 mol%) in unilamellar EYPC vesicles loaded with 500 mM NaCl buffered to pH 7.0 with 25 mM HEPES. The vesicles were dispersed in 25 mM HEPES buffer (pH 7.0) containing 500 mM NaNO3 and 250 mM Na2SO4, respectively.

Though it is reported that 3,5-bis(trifluoromethyl)phenyl squaramide is more active than its 4-(trifluoromethyl)phenyl analog,13f 3,5-bis(trifluoromethyl)phenyl-substituted compound 2 is 5-fold less active than 4-(trifluoromethyl)phenyl-substituted compound 1. This is a likely consequence of the stronger anion-binding affinity of compound 2, which may enforce the formation of tighter complexes with anions and retard the release of the complexed anions. To test this, we measured the binding affinity toward chloride by means of spectrophotometric titrations. As a consequence, compound 2 exhibits 4-fold greater affinity than compound 1 (Tables 1 and S5, and Fig. S15). It is reported that compound 1 acts as a strong anion-selective receptor,12 in particular for sulfate anions. Therefore, we are concerned about whether compounds 1 and 2 exhibit transport selectivity with regard to anions. To test this, we carried out concentration-dependent pH discharge experiments in the presence of sodium salts of different anions (i.e., Cl–, Br–, I–, NO–3 and SO2– 4 ) (Fig. S8-12) and calculated the corresponding EC50 value for each anion. As a consequence, potent transport activity was observed in the presence of NO–3, Cl–, Br– and I–. The similar EC50 values indicate that compounds 1 and 2 show no significant selectivity with respect to those four monovalent anions.20 The slightly higher transport activity for iodide may be due to the relatively high lipophilicity of this anion. Unexpectedly, in the presence of highly hydrophilic sulfate anions, addition of either compound 1 or 2 of varying concentrations even up to 3 mol%, led to only slight increase in the relative fluorescence intensity of pyranine (Figs. 3a and S12). The dosedependent profiles reveal that the changes in the relative fluorescence intensity are not sensitive to the concentrations. In addition, the chloride efflux was significantly inhibited when the internal nitrate was replaced with sulfate anions (Figs. 3b and S13). These results suggest that compounds 1 and 2 exhibit a very low level

of anionophoric activity (if there is any), and thereby rule out the possibility that compounds 1 and 2 are able to efficiently transport sulfate anions across bilayer membranes. It should be noted that, however, because these compounds exhibit much greater binding affinity for sulfate anions than for chloride anions (Table 1), there is another possibility that compounds 1 and 2 are strongly complexed with sulfate anions so that no free host compounds are available for the transport of chloride anions.21 In conclusion, we have successfully synthesized two tripodal squaramide conjugates and fully characterized them on the basis of NMR (1H and 13C) and ESI MS (LR and HR) data. We have measured their transmembrane ionophoric activity by means of pyranine assay and chloride ion selective electrode technique. The data indicate that these two conjugates exhibit potent anionophoric activity.22 Though it is reported that one of them acts as a strong anion receptor, in particular for sulfate anions, these two compounds exhibit no significant selectivity with respect toward the tested monovalent anions and a very low level of activity in the presence of sulfate anions. The present result highlights the finding that a powerful receptor for a given anion may not be necessarily a potent transmembrane transporter for this anion. Further efforts presently in progress are aimed at synthesizing structurally optimized derivatives to carry out a systematic structure-activity relationship (SAR) assessment and to improve the selectivity for anions. These efforts are made with a view toward the design of novel anion transporters for potential biomedical applications.

A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.03. 024.

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Please cite this article in press as: Cai X.-J., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.03.024