Synthesis of a dimeric 3α-hydroxy-7α,12α-diamino-5β-cholan-24-oate conjugate and its derivatives, and the effect of lipophilicity on their anion transport efficacy

Article abstract of DOI:10.1039/c7ob00289k

A dimeric 3α-hydroxy-7α,12α-diamino-5β-cholan-24-oate conjugate and its derivatives having alkyl chains of varying length from methyl to n-pentyl groups on the amido bonds were synthesized and fully characterized on the basis of NMR (¹H and ¹³C) and ESI MS (LR and HR) data. Their transmembrane anion transport activities were investigated in detail by means of a chloride ion selective electrode technique and the pyranine assay. The data indicate that this set of compounds is capable of promoting the transmembrane transport of anions, presumably via an anion exchange process and a mobile carrier mechanism. Detailed kinetic analysis on the data obtained from both chloride efflux and pH discharge experiments reveals that an optimum log:P range may exist for the transport effectiveness in terms of both k₂/K_diss and EC₅₀ values. The present finding highlights the importance of high anionophoric activity in clarifying the effect of lipophilicity on ion-transport effectiveness.

Full text of DOI:10.1039/c7ob00289k

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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Synthesis of dimeric 3α‐hydroxy‐7α,12α‐diamino‐5β‐cholan‐24‐
oate conjugate and its derivatives, and the effect of lipophilicity
on their anion transport efficacy
Received 00th January 20xx,
Accepted 00th January 20xx
Zhi Li a, Yun Chen a, De‐Qi Yuan b and Wen‐Hua Chen *, a
DOI: 10.1039/x0xx00000x
Dimeric 3α‐hydroxy‐7α,12α‐diamino‐5β‐cholan‐24‐oate conjugate and its derivatives having alkyl chains of varying length
from methyl to n‐pentyl groups on the amido bonds were synthesized and fully characterized on the basis of NMR (1H and
13C) and ESI MS (LR and HR) data. Their transmembrane anion transport activities were investigated in detail by means of
chloride ion selective electrode technique and pyranine assay. The data indicate that this set of compounds is capable of
promoting the transmembrane transport of anions, presumably via an anion exchange process and a mobile carrier
mechanism. Detailed kinetic analysis on the data obtained from both chloride efflux and pH discharge experiments reveals
that an optimum logP range may exist for the transport effectiveness in term of both k2/Kdiss and EC50 values. The present
finding highlights the importance of high anionophoric activity in clarifying the effect of lipophilicity on ion‐transport
effectiveness.
www.rsc.org/
1. Introduction
some cases, an optimal logP value has been identified above or
below which the transport activity diminishes. 9, 10
It is known that anion transport across cell membranes plays a
crucial role at some levels in almost every conceivable biochemical
To gain insight into the effect of lipophilicity on the effectiveness of
a given anion transporter series, in a previous study we have
1
operation. Dysfunction in this process may lead to some serious
disorders, such as cystic fibrosis that is caused by the malfunction in
natural chloride ion channels. 2 Therefore, during the past decades,
increasing interest has been attracted in identifying small‐molecule
organic compounds that are capable of efficiently mediating the
transport of anions, in particular chloride anions across lipid bilayer
reported six squaramido‐functionalized bis(choloyl) conjugates A‐F
11, 12
(Fig. 1) that have clogP values ranging from 3.90 to 8.32.
We
have found that the anionophoric activity changes with the
lipophilicity in a concentration‐dependent fashion. Specifically, at
low concentrations the lipophilicity has little effect on the ion
transport activity. When the concentration increases, the effect of
lipophilicity becomes apparent. This finding makes us reasoning
that high anionophoric activity is desired in clarifying the effect of
lipophilicity on ion transport activity. In addition, though the flexible
alkyl chains on the squaramido subunits are not involved in the
recognition of anions, their effect on the interaction of compounds
A‐F with anions may not be ignored.
3
membranes. These so‐called synthetic anion transporters may
4
serve as alternatives for defective natural anion channels. As a
consequence, various non‐peptidic structures have been reported
to exhibit promising anion transport properties. 5
Anion translocation through a lipid membrane is a complex event
involving multiple equilibria and steps.
6
Identifying the key
structural parameter that affects anion transport activity is crucial
to the rational design of effective anion transporters. Given the
amphiphilic nature of phospholipid bilayer membranes, lipophilicity,
widely measured by a logP value (the logarithm of n‐octanol/water
partition coefficient P), is recognized as one of the major structural
O
O
O
O
HN
NH
NH
HN
OH
OH
OH
O
OH
HO
OH
HN
NH
O
7
factors that affect the activity of synthetic anion transporters. It
O
NHR
O
RHN
has been reported that for a given compound series, the anion
R = Me (A), Et (B), n-C3H7 (C), n-C4H9 (D), n-C5H11 (E), n-C6H13 (F)
transport activity may be optimized by varying the lipophilicity. 8 In
R
R
N
N
O
O
a
H2N
NH2
NH2
H2N
Guangdong Provincial Key Laboratory of New Drug Screening, School of
Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, P. R.
HO
OH
China. E‐mail: whchen@smu.edu.cn
Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Minatojima 1‐1‐3,
R = H (1), Me (2), Et (3), n-C3H7 (4), n-C4H9 (5), n-C5H11 (6)
b
Chuo‐ku, Kobe 650‐8586, Japan
† Electronic Supplementary Informaꢀon (ESI) available: Structural characterization
Figure 1. Structures of compounds A‐F and 1‐6.
and measurement of the ion transport activity of compounds
10.1039/x0xx00000x
1‐6. See DOI:
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To test this and also to have a closer look at the impact of was added to release the residual NaCl in the vesicles and the final
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lipophilicity on transport effectiveness, we designed dimeric 3α‐ reading of the electrode was used to calibrDatOeI:t1h0e.1013090/%C7rOeBle0a0s2e89oKf
hydroxy‐7α,12α‐diamino‐5β‐cholan‐24‐oate conjugate 1 linked with chloride. The data are shown in Fig. 2a and S52‐53, and indicate
p‐bis(aminomethyl)benzene, and its derivatives 2‐6 having alkyl that compounds 1‐6 are capable of promoting chloride efflux from
chains of varying length (Fig. 1). Here, the 7‐, and 12‐hydroxyl the EYPC vesicles and the chloride efflux rate is concentration
groups of the choloyl skeleton are transformed into amino dependent.
substituents. This transformation is expected to strengthen the
The results of chloride efflux experiments suggest that compounds
hydrogen‐bonding ability toward anions and thereby lead to an
‐
1‐6 transport chloride by either Cl‐/NO3 antiport or Cl‐/cation
13
enhancement in anion transport activity.
As a matter of facts,
symport mechanism. To distinguish the probable mechanism of
action, we changed the external solution from NaNO3 to Na2SO4
and re‐measured the chloride efflux. The sulfate anion is strongly
such transformation has been demonstrated powerful in the
generation of effective anion receptors and transporters.
Modification of compound 1 with alkyl chains from methyl to n‐
pentyl groups (to give compounds 2‐6), is made on the amido bonds.
This modification should have no effect on the interaction of the 7‐,
and 12‐amino substitutents with anions, but gradually increases the
14
hydrated and is assumed to be unable to readily pass across a lipid
18
bilayer.
If a compound functions as a Cl‐/anion exchanger, its
chloride efflux activity should be inhibited in the presence of sulfate
anions. The results of compounds 1‐6 are shown in Fig. 2b and S54,
and indicate that the chloride efflux rates of compounds 1‐6 were
significantly inhibited by sulfate anions, suggesting that compounds
1‐6 function as anion exchangers.
lipophilicity as the predominant factor that regulates the activity of
15
compounds 1‐6.
Thus, such modification makes it feasible to
assess how lipophilicity is correlated with the transport efficiency of
compounds 1‐6. Herein we report the synthesis of compound 1 and
its derivatives 2‐6 and their transmembrane anionophoric activity
investigated by means of chloride ion selective electrode technique
and pyranine assays. The effect of lipophilicity on the ion transport
activity is discussed in detail.
To gain further insights into the probable mechanism of action and
ion selectivity of compounds 1‐6, we carried out the chloride efflux
experiments in the presence of the chloride salts of group I alkali
metal ions, including Li+, Na+, K+, Rb+ and Cs+. The results are shown
in Fig. 3a and S55 and indicate that the chloride efflux activity of
compounds 1‐6 is essentially independent of group I alkali metal
2. Results and discussion
ions, excluding any meaningful role of those metal ions in the
19
permeation process.
Then, we measured the pH discharge
2.1 Chemistry
activity of compounds 1‐6 in the presence of the sodium salts of
‐
different anions (i.e., NO3 , Cl‐, Br‐, and I‐). In this test, a series of
Compounds 1‐6 were synthesized according to the approach shown
in Scheme 1. Thus, methyl 3α‐acetoxy‐7α,12α‐di[N‐(t‐
EYPC vesicles containing sodium salts and pyranine buffered to pH
7.0 were prepared and suspended in the same sodium salt solution
buffered to pH 8.0. The fluorescence of pyranine was monitored
upon the addition of compounds 1‐6. The results are shown in Fig.
3b and S56 and suggest that compounds 1‐6 are capable of inducing
pH discharge across the membrane. The activity follows the order
of I‐ > NO3‐ ≈ Br‐ ≈ Cl‐, correlating well with a halide transport based
butyloxycarbonyl)amino]‐5β‐cholan‐24‐oate 7 was prepared using
13, 14
literature protocols
and hydrolyzed by LiOH to give 3α‐
hydroxy‐7α,12α‐di[N‐(t‐butyloxycarbonyl)amino]‐5β‐cholan‐24‐oic
acid 8. Activation of compound 8 with N‐hydroxysuccinimide (NHS)
or 1‐hydroxybenzotriazole (HOBt) and subsequent reaction with p‐
bis(aminomethyl)benzene 9 or p‐bis(alkylaminomethyl)benzene 16‐
20 afforded Boc‐protected dimeric conjugates 21‐26. Final
deprotection of the Boc groups in compounds 21‐26 with TFA gave
compounds 1‐6. Compounds 1‐6 were fully characterized on the
basis of NMR (1H and 13C) and ESI MS (LR and HR) data (see
experimental section and Fig. S1‐51).
20
on the lyotropic sequence. These observations imply that pH
gradient decay correlates with OH‐/Cl‐ antiport.
Finally, to determine the mechanism of action of compounds 1‐6,
we measured their pH discharge activity across the lipid
membranes derived from 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐
phosphocholine (POPC) and cholesterol. It is reported that the
addition of cholesterol to the membrane reduces membrane
fluidity, therefore cholesterol assays have been frequently used as
evidence for a mobile carrier mechanism. 21 As shown in Fig. 4 and
S57, the pH discharge activity of compounds 1‐6 was found to be
significantly reduced across the POPC/cholesterol membranes. This
supports a mobile carrier over a channel mechanism.
2.2 Anionophoric activity of compounds 1‐6
To test whether compounds 1‐6 possess anion transport activity,
firstly, we carried out chloride efflux experiments by using chloride
16, 17
ion selective electrode (ISE).
In the experiments, a series of
large unilamellar egg‐yolk phosphatidylcholine (EYPC)‐based
vesicles (100 nm diameter, extrusion) loaded with NaCl were
prepared and suspended in an external isotonic NaNO3 solution. A
sample of compounds 1‐6 (of varying concentration in molar
percent with respect to the total concentration of lipid) was added
as a DMSO solution. The efflux of chloride from the vesicles was
detected by ISE. After 300 s, 5 wt% aqueous Triton X‐100 solution
Taken together, the above‐mentioned observations suggest that
compounds 1‐6 are capable of mediating the transmembrane
transport of chloride via an anion exchange process and a mobile
carrier mechanism.
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O
O
O
O
(iv)
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(i)
(ii)~(iii)
OMe
OH
N
R
1-6
DOI: 10.1039/C7OB00289K
N
R
NHBoc
NHBoc
NHBoc
NHBoc
NHBoc
NHBoc
BocHN
BocHN
AcO
H2N
HO
HO
7
8
OH
R = H (21), Me (22), Et (23), n-C3H7 (24)
n-C4H9 (25), n-C5H11 (26)
(v)
(vi)
(vii)
N
Ns
Ns
N
NH2
HN Ns
NHR
R = Me (16), Et (17),
Ns NH
RHN
R
R
9
10
R = Me (11), Et (12),
n-C3H7 (13), n-C4H9 (14)
n-C5H11 (15)
n-C3H7 (18), n-C4H9 (19)
n-C5H11 (20)
Scheme 1. Synthesis of compounds 1‐6. Reagents and conditions: (i) LiOH, MeOH, reflux; (ii) NHS (or HOBt), DCC, CHCl3 or THF, room
temperature; (iii) p‐bis(aminomethyl)benzene 9 or p‐bis(alkylaminomethyl)benzene 16‐20, Et3N, CHCl3 or THF, room temperature; (iv) TFA,
CH2Cl2, room temperature; (v) p‐nitrobenzene sulfonyl chloride, DMF; (vi) RI (R = Me, Et, n‐C3H7, n‐C4H9, or n‐C5H11), K2CO3, DMF; (vii) 4‐
methoxythiophenol, K2CO3, DMF.
(a)
(b)
Figure 2. (a) Relative chloride efflux promoted by compounds 1‐6 (2 mol%) in EYPC vesicles loaded with 500 mM NaCl buffered to pH 7.0
with 25 mM HEPES. The vesicles were dispersed in 500 mM NaNO3 buffered to pH 7.0 with 25 mM HEPES. (b) Chloride efflux promoted by
compound 1 (5 mol%) in the same 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. The control experiments were
conducted in NaNO3 media and in the absence of compound 1.
(a)
(b)
Figure 3. (a) Relative chloride efflux promoted by compound 1 (5 mol%) in EYPC vesicles loaded with 500 mM MCl (M = Li, Na, K, Rb and Cs)
buffered to pH 7.0 with 25 mM HEPES. The vesicles were dispersed in 500 mM NaNO3 buffered to pH 7.0 with 25 mM HEPES. The control
experiments were conducted in NaCl media with DMSO. (b) Discharge of a pH gradient by compound 1 (0.5 mol%) across EYPC‐based
liposomal membranes, 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) (X = NO3, Cl, Br and I). λEx 460 nm; λem 510 nm. Each profile represents the real
increment in the presence of compound 1.
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the initial rate constants against the concentrations of each of
compounds 1‐6 according to equation (2), gave the Hill coefficient n
26
and the EC50 value of each compound. Here EC50 is defined as
effective transporter loading that needs to reach 50% of the
maximum rate (kmax) after a specified time period and thereby a
measure of the effectiveness of a given transporter. 27
n
Kin = k0 + kmax[monomer]n/([monomer]n + EC50
)
(2)
The kinetic parameters obtained from both analyses are listed in
Table 1. Several observations can be abstracted from these data.
Firstly, compounds 1‐6 have EC50 values of 0.11~0.89 mol%, up to
25‐fold lower than those of compounds A‐F, suggesting that
transformation of the 7‐ and 12‐hydroxyl groups into amino groups
greatly enhances the anionophoric activity. The enhancement in the
transport effectiveness might be ascribed to the following aspects.
Firstly, as already stated in the Introduction, the modification of
compounds A‐F with alkyl chains was made on the squaramido
functional groups, which may affect the interaction with anions. In
contrast, the amino groups in compounds 1‐6 are not substituted,
which would be favorable to the multiple interactions with anions.
Figure 4. Discharge of a pH gradient promoted by compound 1 (1
mol%) across POPC and POPC‐cholesterol (7/3)‐based liposomal
membranes. Measuring conditions for internal vesicles: 0.1 mM
pyranine in 25 mM HEPES (50 mM NaCl, pH 7.0); and for external
vesicles: 25 mM HEPES (50 mM NaCl, pH 8.0). λEx 460 nm; λem 510
nm.
2.3 Effect of lipophilicity on the anionophoric activity of compounds
1‐6
14
Secondly, because both compounds 1‐6 and A‐F function, most
possibly as carriers, 11 the former are smaller in size than the latter
and would be more ready to shuttle back and forth within the
interior of the membranes. In addition, it should be noted that
As shown above, compounds 1‐6 exhibit different chloride efflux
activity, suggesting that lipophilicity has significant effect on the
anion‐transport activity. To evaluate this effect, we firstly measured
compounds 1‐6 are comparable to (thio)urea‐based anion
22
28
the logP values of compounds 1‐6. Specifically, compounds 1‐6
transporters (EC50 = 0.42 mol%),
and much more active than
29
were partitioned in a mixture of n‐octanol and water. After
partition equilibrium was achieved, the concentrations of each of
compounds 1‐6 in organic and aqueous phases were measured by
means of gray value analysis on TLC by ImageJ2X software. The
measured logP values of compounds 1‐6 are listed in Table 1. The
logP values from 0.40 to 0.93 suggest that the lipophilicity of
compounds 1‐6 increases gradually with alkyl chains of varying
length from methyl to n‐pentyl groups.
cholic acid‐based molecular umbrellas (EC50 = 5.7 mol%). Thus,
compounds 1‐6 are considered to be a very effective class of anion
transporters.
Secondly, it is clear that compounds 1‐6 have different k2/Kdiss and
EC50 values. A plot of the k2/Kdiss and 1/EC50 values of compounds 1‐
6 against their logP values shows that the transport effectiveness of
this set of compounds increases with lipophilicity on going from
compound 1 to compound 4, and tend to decrease on going from
compound 4 to compound 6 (Fig. 5). This reveals that an optimum
logP range exists for the transport effectiveness. This may be
Then, we performed detailed analysis on the kinetic data of
compounds 1‐6 obtained from chloride selective electrode
techniques. Analysis of the relationship between the chloride efflux
at 260 s and the concentrations of each of compounds 1‐6
rationalized if the effect of lipophilic/hydrophilic balance on the
30
transport process is taken into account.
Increasing the
23
according to equation (1) afforded two kinetic parameters, i.e.,
lipophilicity is favourable for compounds 1‐6 to partition from
aqueous phases into membranes and leads to an increase in the
transport activity. However, because compounds 1‐6 function via a
mobile carrier mechanism, increasing the lipophilicity may lower
their mobility within the lipid membranes and results in a decrease
in the activity. In a sense, the lipophilic/hydrophilic balance plays a
crucial role in the efficient transport of anions by this set of
compounds.
Hill coefficient n and specificity constant k2/Kdiss. The former
represents the stoichiometry of the transport process, whereas the
latter describes the transporter’s ability to facilitate the diffusion of
a given ion and can be used to measure the specificity of a
transporter for a given anion. Here k2 and Kdiss represent the
intrinsic rate constant and the dissociation constant of the self‐
association process, respectively. 24
kobsd = k0 + k2[monomer]n/Kdiss (1)
The Hill coefficients n of compounds 1‐6 obtained from both
experiments are close to 1, which suggests that they do not
aggregate to function.
Because of the high sensitivity of pyranine assays, we conducted
the concentration‐dependent pH discharge experiments of
compounds 1‐6 (Fig. S58‐59) and calculated the initial rate
3. Conclusions
In conclusion, we have successfully synthesized a dimeric 3α‐
25
constants (kin’s) at each concentration. Nonlinear curve fitting of
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hydroxy‐7α,12α‐diamino‐5β‐cholan‐24‐oate conjugate and its five experimental errors. Detailed kinetic analysis of the effect of
View Article Online
DOI: 10.1039/C7OB00289K
derivatives having varying lipophilicity, and fully characterized them lipophilicity on the ion‐transport activity indicates that this set of
on the basis of NMR (1H and 13C) and ESI MS (LR and HR) data. We compounds has an optimal logP range for the effectiveness in term
have measured their transmembrane ionophoric activity and ion of both k2/Kdiss and EC50. This finding highlights the importance of
selectivity by means of pyranine assay and chloride ion selective high anionophoric activity in addressing the effect of lipophilicity on
electrode technique. The data indicate that these conjugates exhibit transport effectiveness. Further efforts aimed at creating more
potent anionophoric activity across EYPC‐based liposomal effective anion transporters, guided by the present observations,
membranes, presumably via an anion exchange process and a are currently under active investigation in our laboratory. The
mobile carrier mechanism. Of these compounds, the ones with outcome will be reported in due course.
ethyl, n‐propyl and n‐butyl groups display top activity within
Table 1. Kinetic parameters for the chloride efflux and pH discharge by compounds 1‐6
Chloride selective electrode assay b
pH discharge c
Compound
logP a
n
k2/Kdiss (s‐1∙mol%‐1)
0.059±0.004
0.173±0.008
0.320±0.025
0.374±0.026
0.329±0.019
0.297±0.015
n
EC50 (mol%)
0.887±0.016
0.457±0.091
0.115±0.022
0.108±0.011
0.135±0.007
0.138±0.028
1
2
3
4
5
6
0.40±0.021
0.55±0.045
0.64±0.066
0.77±0.048
0.82±0.043
0.93±0.077
0.74±0.06
1.29±0.08
0.68±0.04
0.74±0.06
0.75±0.04
0.77±0.03
1.82±0.18
1.44±0.22
1.13±0.11
1.59±0.14
1.26±0.34
1.38±0.17
a See experimental section for the detailed procedures.
b Measured in EYPC vesicles under the measuring conditions of internal vesicles: 500 mM NaCl in 25 mM HEPES (pH 7.0) and external vesicles: 500 mM NaNO3 in 25
mM HEPES (pH 7.0) (Fig. S53).
c Measured in EYPC vesicles under the measuring conditions of internal vesicles: 0.1 mM pyranine in 25 mM HEPES (50 mM NaCl, pH 7.0) and external vesicles: 25 mM
HEPES (50 mM NaCl, pH 8.0) (Fig. S59). The average rate constant for the background (k0) was (6.46±3.10)×10‐3 min‐1.
made by use of iodine, UV (254 or 365 nm) and 20% aqueous H2SO4.
Liposomes were prepared by extrusion using an Avanti’s Mini‐
Extruder (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA).
Nuclepore track‐etched polycarbonate membranes (100 nm) were
obtained from Whatman (Florham Park, New Jersey, USA). Chloride
efflux was measured on a Mettler‐Toledo PerfectIonTM chloride ion
selective electrode. Fluorescence spectra were measured on a PE
LS55 spectrofluorimeter.
EYPC, POPC and pyranine were purchased from Sigma Chemical Co.
(St Louis, USA). Compound 7 was synthesized starting from cholic
13, 14
acid according to reported protocols.
All the other chemicals
and reagents were obtained from commercial sources and used
without further purification. Buffer solutions were prepared in triply
distilled deionized water.
■
( ) and k2/Kdiss (★) values against the
Figure 5. Plots of the 1/EC50
logP of compounds 1‐6.
Synthesis of compound 8
To a solution of compound 7 (200 mg, 0.32 mmol) in methanol (3.5
mL) was added lithium hydroxide aqueous solution (0.2 mol/L, 3
mL). The reaction was conducted under reflux and monitored by
TLC (CH2Cl2/CH3OH = 30/1, v/v). After 4 h, the reaction mixture was
concentrated under reduced pressure, dissolved with EtOAc (150
mL) and washed with pH 3 HCl aqueous solution (100 mL × 3). The
organic layer was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The obtained residues were purified by
chromatography on a silica gel column, eluted with a mixture of
CH2Cl2 and CH3OH (85/1, v/v) to afford compound 8 (116 mg, 63%)
as a white foam having 1H‐NMR (400 MHz, CD3OD): δ 3.97‐3.95 (m,
1H), 3.57 (br, 1H), 3.50‐3.42 (m, 1H), 2.41‐2.17 (m, 2H), 1.95‐1.12
(m, 40H), 0.97‐0.96 (m, 6H), 0.84 (s, 3H) ppm; 13C‐NMR (100 MHz,
4. Experimental
Generals. 1H and 13C NMR spectra were recorded using a Bruker
Avance AV 400 spectrometer and the deuterium solvents as
standards. LR and HR ESI mass spectra were measured on Waters
UPLC/Quattro Premier XE and Bruker maXis 4G ESI‐Q‐TOF mass
spectrometers, respectively. LR and HR EI mass spectra were
measured on a Thermo DSQ and Thermo MAT 95XP mass
spectrometers, respectively. Silica gel 80‐100 Å (reagent pure,
Qingdao Haiyang Chemical Co. Ltd) was used for column
chromatography. Analytical thin‐layer chromatography (TLC) was
performed on silica gel plates 60 GF254. Detection on TLC was
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Journal Name
CD3OD): δ 176.8, 156.5, 78.4, 71.0, 53.3, 48.3, 44.5, 43.8, 41.4, 38.0, δ 8.36 (d, J = 8.8 Hz, 4H), 8.00 (d, J = 8.8 Hz, 4H), 7.25 (Vsi,ew4HAr)t,ic4le.3O7nli(nse,
36.5, 34.8, 34.7, 34.0, 31.3, 30.7, 30.6, 29.3, 28.4, 27.5, 27.4, 27.0, 4H), 3.15 (t, J = 7.6 Hz, 4H), 1.36‐1.28 (m,D4OHI:),101..11093‐91/.C079O(Bm00,248H9)K,
26.7, 22.6, 21.6, 16.8, 12.3 ppm; negative ESI‐MS: m/z 605.8 ([M‐H]‐) 0.77 (t, J =7.2 Hz, 6H) ppm; EI MS: m/z 618.19 (M+) and HR‐EI‐MS
and HR‐ESI‐MS for C34H59O7N2 ([M+H]+) Calcd: 607.43168; Found: for C28H34O8N4S2 (M+) Calcd: 618.1813; Found: 618.1805.
607.43188.
Compound 15. Procedure as described for 11; from 10 (300 mg,
0.59 mmol) and 1‐iodopentane (1.55 mL, 11.86 mmol). Yield: 236
mg (62%), a yellow powder. Mp 226‐227 °C; H‐NMR (400 MHz,
1
Synthesis of compound 10
CDCl3): δ 8.36 (d, J = 8.8 Hz, 4H), 8.00 (d, J = 8.8 Hz, 4H), 7.25 (s, 4H),
4.37 (s, 4H), 3.14 (t, J = 7.6 Hz, 4H), 1.37‐1.30 (m, 4H), 1.21‐1.04 (m,
8H), 0.78 (t, J = 7.2 Hz, 6H) ppm; EI MS: m/z 646.21 (M+) and HR‐EI‐
MS for C30H38O8N4S2 (M+) Calcd: 646.2126; Found: 646.2136.
To a solution of p‐bis(aminomethyl)benzene 9 (500 mg, 3.67 mmol)
in DMF (4.5 mL) was added slowly a solution of p‐nitrobenzene
sulfonyl chloride (2.44 g, 11.01 mmol) in DMF (9.5 mL). The reaction
mixture was stirred at room temperature. The reaction was
monitored with TLC (CH2Cl2/CH3OH = 35/1, v/v). After 12 h, the
reaction mixture was added to saturated sodium bicarbonate
aqueous solution (250 mL) with vigorous stirring. The resulting
yellow precipitates were collected by filtration and washed
thoroughly with water to afford compound 10 (965 mg, 52%) as a
pale yellow powder. Mp 194‐195 °C; 1H‐NMR (400 MHz, DMSO‐d6):
δ 8.32 (d, J = 8 Hz, 4H), 7.95 (d, J = 8 Hz, 4H), 7.09 (s, 4H), 3.95 (s, 4H)
ppm; negative ESI‐MS: m/z 505.3 ([M‐H]‐) and negative HR‐ESI‐MS
for C20H17O8N4S2 ([M‐H]‐) Calcd: 505.04933; Found: 505.04941.
Syntheses of compounds 21‐26
Compound 21. To a solution of 8 (116 mg, 0.19 mmol) and NHS (43
mg, 0.37 mmol) in CHCl3 (6 mL) was added DCC (134 mg, 0.65
mmol). The resulting mixture was stirred at room temperature. The
reaction was monitored by TLC (CH2Cl2/CH3OH = 25/1, v/v). After
5.5 h, a solution of p‐bis(aminomethyl)benzene 9 (11 mg, 0.081
mmol) in CHCl3 (2.5 mL) was added. The resulting mixture was
stirred at room temperature. The reaction was monitored with TLC
(CH2Cl2/CH3OH = 18/1, v/v). After 24 h, the reaction mixture was
diluted with CHCl3 (150 mL) and washed subsequently with
saturated NaHCO3 (150 mL × 2), water (150 mL) and saline (150 mL).
The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The obtained residues were
purified by chromatography on a silica gel column, eluted with a
mixture of CH2Cl2 and CH3OH (40/1, v/v) to afford compound 21 (59
mg, 56% based on compound 9) as a white solid. Mp 157‐158 °C;
1H‐NMR (400 MHz, CDCl3/CD3OD, 10/1, v/v): δ 7.22 (s, 4H), 4.36 (s,
4H), 3.92 (br, 2H), 3.57 (br, 2H), 3.36 (br, 2H), 2.32‐0.93 (m, 92H),
0.90 (s, 6H), 0.77 (s, 6H) ppm; 13C‐NMR (100 MHz, CDCl3/CD3OD,
10/1, v/v): δ 174.5, 156.3, 137.4, 127.7, 78.6, 78.5, 77.4, 71.2, 52.8,
47.2, 44.8, 43.8, 42.9, 41.3, 38.0, 36.5, 35.0, 34.9, 34.2, 33.6, 31.7,
31.6, 29.5, 28.5, 28.3, 28.2, 27.0, 23.0, 22.4, 17.6, 13.2 ppm; ESI‐MS:
m/z 1335.7 ([M+Na]+) and negative HR‐ESI‐MS for C76H123O12N6
([M–H]–) Calcd: 1311.92045; Found: 1311.91748.
Syntheses of compounds 11‐15
Compound 11. A mixture of compound 10 (250 mg, 0.49 mmol) and
anhydrous potassium carbonate (684 mg 4.94 mmol) in anhydrous
DMF (9 mL) was stirred at room temperature for 30 min. Then
methyl iodide (1.5 mL, 9.88 mmol) was added. The resulting
mixture was stirred at room temperature. The reaction was
monitored with TLC (petroleum ether/ CH2Cl2 = 1/15, v/v). After 40
h, the reaction mixture was added to water (25 mL). The formed
yellow precipitates were collected by filtration, washed thoroughly
with water and dried in vacuum to afford compound 11 (156 mg,
59%) as a yellow powder. Mp 205‐206 °C; 1H‐NMR (400 MHz,
DMSO‐d6): δ 8.45 (d, J = 7.6 Hz, 4H), 8.12 (d, J = 7.6 Hz, 4H), 7.31 (s,
4H), 4.22 (s, 4H), 2.62 (s, 6H) ppm; EI MS: m/z 534.09 (M+) and HR‐
EI‐MS for C22H22O8N4S2 ([M]+) Calcd: 534.0874; Found: 534.0866.
Compound 12. Procedure as described for 11; from 10 (500 mg,
0.99 mmol) and ethyl iodide (1.45 mL, 19.76 mmol). Yield: 365 mg
(66%), a yellow powder. Mp 209‐210 °C; 1H‐NMR (400 MHz, CDCl3):
δ 8.36 (d, J = 8.4 Hz, 4H), 8.01 (d, J = 8.4 Hz, 4H), 7.27 (s, 4H), 4.39 (s,
4H), 3.28‐3.23 (m, 4H), 0.97 (t, 6H, J = 7.2 Hz) ppm; EI MS: m/z
562.12 (M+) and HR‐EI‐MS for C24H26O8N4S2 (M+) Calcd: 562.1187;
Found: 562.1180.
Compound 22. A solution of compound 11 (250 mg, 0.47 mmol) and
potassium carbonate (324 mg, 2.34 mmol) in DMF (2 mL) was
stirred at room temperature for 30 min. Then 4‐methoxythiophenol
(460 μL, 3.75 mmol) was added. The reaction mixture was stirred at
room temperature. The reaction was monitored with TLC
(CH3OH/NH3∙H2O = 30/1, v/v). After 9.5 h, the reaction mixture was
diluted with CHCl3 (150 mL) and washed with saturated Na2CO3
(100 mL × 3). The organic layer was dried over anhydrous Na2SO4.
Purification was achieved by flash chromatography on a silica gel
column, eluted with a mixture of CH3OH and NH3∙H2O (100/1, v/v)
to give compound 16 (42 mg, 55%). The removal of 4‐
nitrobenzenesulfonyl groups was confirmed with ESI‐MS: m/z 165.5
([M+H]+). Because of the potential sensitivity to oxidation,
compound 16 was used in the next step without further
characterization. Then, to a solution of compound 8 (150 mg, 0.25
mmol) and HOBt (100 mg, 0.74 mmol) in anhydrous THF (3.5 mL)
was added DCC (179 mg, 0.87 mmol). The resulting mixture was
stirred at room temperature. The reaction was monitored by TLC
Compound 13. Procedure as described for 11; from 10 (500 mg,
0.99 mmol) and 1‐iodopropane (1.90 mL, 19.76 mmol). Yield: 355
1
mg (61%), a yellow powder. Mp 214‐215 °C; H‐NMR (400 MHz,
CDCl3): δ 8.36 (d, J = 8.4 Hz, 4H), 8.00 (d, J = 8.4 Hz, 4H), 7.25 (s, 4H),
4.37 (s, 4H), 3.12 (t, J = 7.6 Hz, 4H), 1.39‐1.34 (m, 4H), 0.73 (t, J =7.6
Hz, 6H) ppm; EI MS: m/z 590.15 (M+) and HR‐EI‐MS for C26H30O8N4S2
(M+) Calcd: 590.1500; Found: 590.1507.
Compound 14. Procedure as described for 11; from 10 (300 mg,
0.59 mmol) and 1‐iodobutane (1.35 mL, 11.86 mmol). Yield: 230 mg
(63%), a yellow powder. Mp 220‐221 °C; 1H‐NMR (400 MHz, CDCl3):
6 | J. Name., 2012, 00, 1‐3
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Organic & Biomolecular Chemistry
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ARTICLE
(CH2Cl2/CH3OH = 25/1, v/v). After 5.5 h, a solution of compound 16 (m, 6H), 2.38‐2.11 (m, 4H), 1.81‐0.80 (m, 106H), 0.71‐0.66 (m, 6H)
View Article Online
13
(18 mg, 0.11 mmol) and Et3N (0.5 mL) in anhydrous THF (440 μL) ppm; C‐NMR (100 MHz, CDCl3/CD3OD, 10D/1O,I:v1/0v.)1:0δ391/C774O.3B,0105268.94K,
was added to the above reaction mixture. The resulting mixture was 137.4, 136.9, 128.5, 128.2, 127.0, 126.6, 78.8, 78.7, 71.5, 53.0, 51.1,
stirred at room temperature. The reaction was monitored with TLC 48.1, 47.4, 46.1, 45.0, 44.1, 41.5, 38.3, 36.8, 35.4, 35.1, 34.5, 31.8,
(CH2Cl2/CH3OH = 20/1, v/v). After 14 h, the reaction mixture was 30.8, 30.5, 29.8, 29.7, 28.6, 28.5, 27.4, 27.2, 23.2, 22.7, 20.3, 20.2,
diluted with CHCl3 (150 mL) and washed with saturated NaHCO3 18.0, 13.8, 13.5 ppm; ESI‐MS: m/z 1447.9 ([M+Na]+) and HR‐ESI‐MS
(150 mL × 2). The organic layer was dried over anhydrous Na2SO4 for C84H141O12N6 ([M+H]+) Calcd: 1426.0602; Found: 1426.06018.
and concentrated under reduced pressure. The obtained residues
Compound 26. Compound 20 was prepared using the procedures as
described for compound 16. The removal of 4‐nitrobenzenesulfonyl
group was confirmed with ESI‐MS: ESI‐MS: m/z 277.7 ([M+H]+). The
rest procedure as described for 16; from 8 (125 mg, 0.21 mmol) and
20 (26 mg, 0.093 mmol). Yield: 58 mg (44%), a white solid. Mp 169‐
170 °C; 1H‐NMR (400 MHz, CD3Cl/CD3OD, 10/1,v/v): δ 7.16‐7.04 (m,
4H), 4.52‐4.45 (m, 4H), 3.91‐3.87 (m, 2H), 3.54 (br, 2H), 3.47‐3.37
(m, 2H), 3.23‐3.09 (m, 4H), 2.38‐2.16 (m, 4H), 1.85‐0.70 (m, 116H)
ppm; 13C‐NMR (100 MHz, CDCl3/CD3OD, 10/1, v/v): δ 174.3, 156.4,
137.4, 136.9, 128.5, 128.2, 127.0, 126.6, 78.8, 71.5, 53.0, 51.1, 48.1,
47.4, 46.3, 45.0, 44.1, 41.5, 38.3, 36.8, 35.4, 35.1, 34.5, 31.8, 30.5,
29.8, 29.2, 29.1, 28.7, 28.6, 28.5, 27.4, 27.1, 23.2, 22.7, 22.5, 22.4,
were purified by preparative TLC (petroleum ether/EtOAc = 1/2, v/v)
to afford compound 22 (99 mg, 67%) as a white solid. Mp 160‐
1
161 °C; H‐NMR (400 MHz, CD3OD): δ 7.30‐7.18 (m, 4H), 4.64‐4.59
(m, 4H), 3.96‐3.91 (m, 2H), 3.57 (br, 2H), 3.48‐3.42 (m, 2H), 3.00‐
2.95 (m, 6H), 2.53‐2.30 (m, 4H), 1.95‐0.80 (m, 98H) ppm; 13C‐NMR
(100 MHz, CD3OD): δ 174.7, 156.5, 136.4, 136.1, 128.1, 127.7, 126.7,
126.3, 78.4, 70.9, 53.1, 53.0, 50.1, 44.6, 43.7, 41.4, 38.1, 36.5, 35.0,
34.8, 34.7, 34.3, 34.0, 31.3, 31.0, 30.1, 29.4, 28.4, 27.5, 27.4, 26.9,
26.7, 22.7, 21.6, 17.1, 12.4 ppm; ESI‐MS: m/z 1363.6 ([M+Na]+) and
HR‐ESI‐MS for C78H129O12N6 ([M+H]+) Calcd: 1341.9663; Found:
1341.96655.
Compound 23. Compound 17 was prepared using the procedures as 18.0, 14.0, 13.5 ppm; ESI‐MS: m/z 1475.9 ([M+Na]+) and HR‐ESI‐MS
described for compound 16. The removal of 4‐nitrobenzenesulfonyl for C86H145O12N6 ([M+H]+) Calcd: 1454.0915; Found: 1454.09412.
group was confirmed with ESI‐MS: m/z 193.4 ([M+H]+). The rest
procedure as described for 22; from 8 (150 mg, 0.25 mmol) and 17
Syntheses of compounds 1‐6
(21 mg, 0.11 mmol). Yield: 86 mg (57%), a white solid. Mp 162‐
1
Compound 1. To a solution of 21 (49 mg, 0.037 mmol) in CH2Cl2 (2
mL) was added TFA (0.57 mL, 7.42 mmol). The resulting mixture was
stirred at room temperature. The reaction was monitored with TLC
(CH2Cl2/CH3OH = 18/1, v/v). After 4 h, the reaction mixture was
evaporated under reduced pressure. The obtained yellow residues
were dissolved in MeOH (0.4 mL) and transferred to a centrifuging
tube. Ammonia solution (25%, 12 mL) was then added and the
resulting mixture was centrifuged for 10 min. The clear upper
solution was disposed and water (12 mL) was added. The resulting
mixture was re‐centrifuged for 5 min and the clear upper solution
was disposed. The obtained precipitates were dissolved in CH3OH (1
mL) and concentrated under reduced pressure. The obtained
residue was purified by chromatography on a silica gel column,
eluted with a mixture of CH3OH and NH3∙H2O (150/1, v/v) to give
163 °C; H‐NMR (400 MHz, CD3OD): δ 7.29‐7.18 (m, 4H), 4.64‐4.58
(m, 4H), 3.96‐3.88 (m, 2H), 3.57 (br, 2H), 3.48‐3.36 (m, 6H), 2.48‐
2.26 (m, 4H), 1.95‐0.80 (m, 104H) ppm; 13C‐NMR (100 MHz,
CDCl3/CD3OD, 10/1, v/v): δ 174.1, 156.2, 156.0, 137.1, 136.6, 128.2,
127.9, 126.8, 126.4, 78.6, 71.3, 52.8, 50.8, 48.9, 47.9, 47.1, 45.8,
44.8, 43.9, 41.3, 38.0, 36.6, 35.1, 34.9, 34.2, 31.6, 30.6, 29.5, 28.3,
28.2, 27.2, 26.9, 23.0, 22.4, 19.9, 17.8, 13.6, 13.2 ppm; ESI‐MS: m/z
1391.7 ([M+Na]+) and HR‐ESI‐MS for C80H133O12N6 ([M+H]+) Calcd:
1369.9976; Found: 1369.99915.
Compound 24. Compound 18 was prepared using the procedures as
described for compound 16. The removal of 4‐nitrobenzenesulfonyl
group was confirmed with ESI‐MS: m/z 221.8 ([M+H]+). The rest
procedure as described for 22; from 8 (150 mg, 0.25 mmol) and 18
(25 mg, 0.11 mmol). Yield: 114 mg (72%), a white solid. Mp 165‐
1
compound 1 (23 mg, 67%) as a yellow solid. Mp 117‐118 °C; H‐
1
166 °C; H‐NMR (400 MHz, CD3OD): δ 7.29‐7.17 (m, 4H), 4.66‐4.56
NMR (400 MHz, CD3OD): δ 7.25 (s, 4H), 4.35 (s, 4H), 3.43 (br, 2H),
3.31 (br, 2H), 3.17 (br, 2H), 2.35‐1.14 (m, 56H), 1.05 (s, 6H), 0.98 (s,
6H) , 0.81 (s, 6H) ppm; 13C‐NMR (100 MHz, CD3OD): δ 174.9, 137.7,
127.3, 71.0, 54.3, 45.5, 42.3, 41.3, 38.9, 38.3, 35.3, 34.9, 34.5, 32.7,
32.6, 31.6, 29.7, 27.4, 25.9, 25.8, 22.9, 21.3, 16.1, 12.3 ppm; ESI‐MS:
m/z 914.4 ([M+H]+) and negative HR‐ESI‐MS for C56H91O4N6 ([M–H]–)
Calcd: 911.71073; Found: 911.7078.
(m, 4H), 3.96‐3.90 (m, 2H), 3.57 (br, 2H), 3.48‐3.40 (m, 2H), 3.29‐
3.24 (m, 4H), 2.48‐2.27 (m, 4H), 1.95‐0.80 (m, 108H) ppm; 13C‐NMR
(100 MHz, CDCl3/CD3OD, 10/1, v/v): δ 174.1, 156.1, 137.1, 136.6,
128.2, 128.0, 126.7, 126.4, 78.6, 71.3, 52.8, 50.8, 47.9, 47.1, 46.1,
44.8, 43.9, 41.3, 38.0, 36.6, 35.2, 34.9, 31.6, 30.3, 29.5, 29.0, 28.8,
28.5, 28.3, 28.2, 27.2, 26.9, 23.0, 22.4, 22.2, 17.8, 13.7, 13.3 ppm;
ESI‐MS: m/z 1420.0 ([M+Na]+) and HR‐ESI‐MS for C82H137O12N6
([M+H]+) Calcd: 1398.0289; Found:1398.03052.
Compound 2. To a solution of 22 (83 mg, 0.062 mmol) in CH2Cl2 (2
mL) was added TFA (0.95 mL, 12.30 mmol). The resulting solution
was stirred at room temperature. The reaction was monitored with
TLC (CH2Cl2/CH3OH = 15/1, v/v). After 5.5 h, the reaction mixture
was evaporated under reduced pressure. The obtained yellow
residues were dissolved in MeOH (0.4 mL) and added into ammonia
solution (25%, 18 mL). The resulting yellowish precipitates were
collected by filtration and re‐dissolved in MeOH. The MeOH
solution was evaporated under reduced pressure to afford 2 (34 mg,
Compound 25. Compound 19 was prepared using the procedures as
described for compound 16. The removal of 4‐nitrobenzenesulfonyl
group was confirmed with ESI‐MS: m/z 249.8 ([M+H]+). The rest
procedure as described for 22; from 8 (162 mg, 0.27 mmol) and 19
(30 mg, 0.12 mmol). Yield: 69 mg (40%), a white solid. Mp 168‐
169 °C; 1H‐NMR (400 MHz, CDCl3/CD3OD, 10/1, v/v): δ 7.13‐7.00 (m,
4H), 4.48‐4.42 (m, 4H), 3.87‐3.83 (m, 2H), 3.50 (br, 2H), 3.27‐3.06
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1‐3 | 7
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59%) as a yellow solid. Mp 121‐122 °C; 1H‐NMR (400 MHz, CD3OD): water‐saturated n‐octanol and n‐octanol‐saturated water in equal
View Article Online
DOI: 10.1039/C7OB00289K
δ 7.30‐7.19 (m, 4H), 4.69‐4.59 (m, 4H), 3.46‐3.41 (m, 2H), 3.21‐3.16 volume (0.5 mL each) and allowed to equilibrate at room
(m, 2H), 3.05‐2.96 (m, 8H), 2.58‐2.38 (m, 4H), 2.19‐0.79 (m, 62H) temperature over 10 h. The aqueous and organic phases were
ppm; 13C‐NMR (100 MHz, CD3OD): δ 175.2, 174.7, 136.8, 136.5, separated, and aliquots (5.0 μL) of each phase were deposited onto
128.0, 127.7, 126.8, 126.4, 71.1, 54.1, 52.8, 50.1, 45.8, 41.6, 41.5, a thin layer chromatography plate, dried and stained with ninhydrin
39.2, 39.0, 35.4, 34.9, 34.6, 34.3, 33.5, 33.3, 30.9, 30.0, 29.8, 27.4, alcohol solution (2%, wt). The plate was scanned and transformed
26.4, 25.8, 23.0, 21.5, 16.2, 12.5 ppm; ESI‐MS: m/z 941.7 ([M+H]+) into gray. The density of each spot was analyzed by gray value
and HR‐ESI‐MS for C58H97O4N6 ([M+H]+) Calcd: 941.75658; Found: analysis of ImageJ2X software. The ratio of the gray values from
941.75702.
organic and aqueous phases represents the oil‐water partition
coefficient P. Experiments were repeated twenty times and the
average values were taken.
Compound 3. Procedure as described for 2; from 23 (88 mg, 0.064
mmol). Yield: 37 mg (59 %), a yellow solid. Mp 122‐123 °C; 1H‐NMR
(400 MHz, CD3OD): δ 7.30‐7.19 (m, 4H), 4.67‐4.60 (m, 4H), 3.52‐3.37
(m, 6H), 3.19‐3.14 (m, 2H), 3.03 (br, 2H), 2.56‐2.29 (m, 4H), 2.21‐
0.78 (m, 68H) ppm; 13C‐NMR (100 MHz, CD3OD): δ 174.7, 174.5,
137.3, 136.9, 127.9, 127.6, 126.7, 126.3, 71.1, 54.1, 50.4, 45.9, 41.7,
41.6, 39.3, 39.1, 35.4, 34.9, 34.6, 33.6, 31.5, 31.2, 29.8, 29.5, 29.3,
27.4, 26.5, 25.8, 23.0, 22.3, 21.6, 16.3, 12.6, 11.5 ppm; ESI‐MS: m/z
969.7 ([M+H]+) and HR‐ESI‐MS for C60H101O4N6 ([M+H]+) Calcd:
969.78788; Found: 969.78821.
Measurement of chloride efflux and pH discharge activity
The preparation of vesicles and the measurement of the chloride
efflux and pH discharge activity of compounds 1‐6 were conducted
using the protocols previously described by us and others. 16, 17
Acknowledgements
This work was financially supported by the National Natural Science
Foundation of China (No. 21072090) and the Program for New
Century Excellent Talents in University (NCET‐11‐0920). WHC
acknowledges the support from the Hyogo Overseas Research
Network (HORN) program, Japan.
Compound 4. Procedure as described for 2; from 24 (93 mg, 0.067
mmol). Yield: 48 mg (72%), a yellow solid. Mp 123‐124 °C; 1H‐NMR
(400 MHz, CD3OD): δ 7.29‐7.18 (m, 4H), 4.68‐4.60 (m, 4H), 3.45‐3.39
(m, 2H), 3.31‐3.22 (m, 6H), 3.12 (br, 2H), 2.55‐2.30 (m, 4H), 2.21‐
0.79 (m, 72H) ppm; 13C‐NMR (100 MHz, CD3OD): δ 175.0, 174.6,
137.1, 136.8, 127.9, 127.6, 126.7, 126.2, 71.0, 54.1, 50.9, 49.2, 48.1,
45.7, 41.4, 39.1, 38.7, 35.4, 34.8, 34.6, 33.1, 31.4, 31.2, 29.7, 29.6,
27.4, 26.2, 25.8, 23.0, 21.5, 21.4, 20.3, 16.2, 12.4, 10.2 ppm; ESI‐MS:
m/z 997.8 ([M+H]+) and HR‐ESI‐MS for C62H105O4N6 ([M+H]+) Calcd:
997.81918; Found: 997.8183.
Notes and references
1
(a) T. J. Jentsch, V. Stein, F. Weinreich and A. A. Zdebik, Physiol.
Rev., 2002, 82, 503‐568; (b) S. K. Ko, S. K. Kim, A. Share, V. M.
Lynch, J. Park, W. Namkung, W. Van Rossom, N. Busschaert, P.
A. Gale, J. L. Sessler and I. Shin, Nat. Chem., 2014, 6, 885‐892.
(a) C. Higgins, Nature, 1992, 82, 536‐536; (b) J. Y. Choi, D.
Muallem, K. Kiselyov, M. G. Lee, P. J. Thomas and S. Muallem,
Nature, 2001, 410, 94‐97.
Compound 5. Procedure as described for 2; from 25 (64 mg, 0.045
mmol). Yield: 29 mg (63%), a yellow solid. Mp 127‐128 °C; 1H‐NMR
(400 MHz, CD3OD): δ 7.30‐7.18 (m, 4H), 4.69‐4.60 (m, 4H), 3.49‐3.37
(m, 4H), 3.31‐3.17 (m, 4H), 3.06 (br, 2H), 2.52‐2.32 (m, 4H), 2.19‐
0.78 (m, 76H) ppm; 13C‐NMR (100 MHz, CD3OD): δ 174.9, 174.6,
137.2, 136.9, 127.9, 127.6, 126.7, 126.3, 71.1, 54.1, 50.8, 46.2, 45.8,
41.6, 39.2, 38.9, 35.4, 34.9, 34.6, 33.5, 31.5, 31.2, 30.4, 29.8, 29.7,
29.3, 27.4, 26.4, 25.8, 23.0, 21.5, 19.8, 19.6, 16.3, 12.8, 12.5 ppm;
ESI‐MS: m/z 1026.0 ([M+H]+) and HR‐ESI‐MS for C64H109O4N6
([M+H]+) Calcd: 1025.85048; Found: 1025.85156.
2
3
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Compound 6. Procedure as described for 2; from 26 (62 mg, 0.043
mmol). Yield: 29 mg (65%), a yellow solid. Mp 129‐130 °C; 1H‐NMR
(400 MHz, CD3OD): δ 7.30‐7.19 (m, 4H), 4.68‐4.60 (m, 4H), 3.46‐3.38
(m, 4H), 3.29‐3.23 (m, 4H), 3.13 (br, 2H), 2.52‐2.30 (m, 4H), 2.21‐
0.79 (m, 80H) ppm; 13C‐NMR (100 MHz, CD3OD): δ 174.9, 174.6,
137.2, 136.9, 127.9, 127.6, 126.7, 126.3, 71.1, 54.1, 50.8, 46.4, 45.7,
41.4, 39.1, 38.6, 35.5, 35.3, 34.8, 34.5, 33.1, 31.5, 29.7, 29.3, 28.8,
28.6, 28.0, 27.4, 26.8, 26.2, 25.8, 23.0, 22.0, 21.4, 16.2, 12.9, 12.4
ppm; ESI‐MS: m/z 1053.9 ([M+H]+) and HR‐ESI‐MS for C66H113O4N6
([M+H]+) Calcd: 1053.88178; Found: 1053.87866.
4
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Partitioning between 1‐octanol and water
This experiment was conducted according to reported protocols. 22
Specifically, a given compound (1 mg) was partitioned between
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Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
Deng, Y. Chen, T. Wu and W.‐H. Chen, Bioorg. Med. Chem. Lett.,
2016, 26, 3665‐3668.
though changes in the composition of the lipid bilayers affect
the rate of transport, the optimum clogP (= 5~6) for peak
View Article Online
DOI: 10.1039/C7OB00289K
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