Impact of Tetracationic Calix[4]arene Conformation—from Conic Structure to Expanded Bolaform—on Their Antibacterial and Antimycobacterial Activities

Article abstract of DOI:10.1002/cbic.201800609

The four possible conformers of a new tetrakisguanidino calix[4]arene thought to interact deleteriously with bacterial membranes have been synthesized, characterized, and evaluated for their in vitro cytotoxicity and antibacterial activity against various

Full text of DOI:10.1002/cbic.201800609

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Accepted Article
Title: Impact of tetracationic calix[4]arene conformation - from conic
structure to expanded bolaform- on their antibacterial and
antimycobacterial activities
Authors: Maxime Mourer, Raphaël Emmanuel Duval, Patricia
Constant, Mamadou Daffé, and Jean-Bernard Regouf de
Vains
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To be cited as: ChemBioChem 10.1002/cbic.201800609
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Impact of tetracationic calix[4]arene conformation - from conic structure
to expanded bolaform - on their antibacterial and antimycobacterial
activities.
Maxime Mourera*, Raphaël E. Duvala,c, Patricia Constantb, Mamadou Dafféb, Jean-Bernard Regnouf-de-
Vainsa
Abstract: The four possible conformers of a new tetra-guanidino
calix[4]arene thought to interact deleteriously with bacterial
membrane were synthesized, characterized and evaluated for their in
vitro cytotoxicity and antibacterial activity against various reference
Gram negative and Gram positive bacteria as well as M. tuberculosis.
It appears that the reversal of at least one phenolic unit allows a clear
increase of the activities. This can be attributed to the evolution
towards bolaform structures prone to interact deeper with the bacterial
membrane. Indeed, the 1,3-alternate conformer 16 exhibits the best
antibacterial activity (MIC < 1.0 µgꞏmL-1 on S. aureus). Moreover, 16
displays very good antibacterial activities against isoniazid-resistant
strain of M. tuberculosis (MIC = 1.2 µgꞏmL-1), associated to the lowest
cytotoxicity, making it the most potent compound of the series; this
can open new ways of research in the field of anti-infective drugs
development to face the huge current demand.
health today".[5] Facing a possible therapeutic impasse due to the
lake of new anti-infective agents, leads to turn to the search of
new anti-infective compounds, aiming to cope with resistance
mechanisms developed by bacteria, regarding the active
compounds commonly used, or to try to face lake of new anti-
infectious agents possessing an innovative mechanism of action.
In this sense, it has been shown that some calixarene derivatives
could be potent and promising antibacterial agents.[3] In this
context, our team is conducting, a program dedicated to the
search for new antibacterial compounds, notably based on
polycationic calixarenes. Our mechanistic hypothesis is based on
deleterious interactions that could occur between the negatively
charged surfaces of bacteria, and the constrained positive
charges introduced on a spatial organizer such as a calixarene
macrocyclic platform. Our first studies were extremely
encouraging with the development, among others, of conic
calixarene structures exhibiting four guanidinium functions on the
upper rim.[6]
Introduction
Owing to the aforementioned organizing behavior, the
introduction of positive charges on the calix[4]arene core results
in an excellent antibacterial agent contrary to its monomeric
constitutive phenolic analogue.[6-9] Afterward we also developed
The last thirty years have shown a growing interest for calixarene
chemistry due to their excellent organizational behavior of various
chemical functions that permits to address very diverse fields of
application.[1] Even if they remain somewhat confidential, the
applications of calixarenes in biology are constantly increasing in
very different fields such as protein-surface recognition, cell
transfection, DNA condensation….[2], as anti-infectives.[3] or as
anti-infectives including intrinsically active species and
prodrugs[3a], but also recent amphiphilic species cumulating both
transfective and antibacterial properties.[3b]
polycationic compounds organized around a benzene core
[10]
(mono-, bis-, tetra- and hexa-guanidinium)
or at the ends of
alkyl chains (bis-guanidinium) that can be considered as flexible
linear organizing templates.[11] Many of these structures showed
excellent activities against both sensitive or resistant bacteria
sometimes with Minimum Inhibitory Concentrations (MIC) < 1
g/mL.[5-9]
In parallel, we started to explore the activity of these compounds
on a particular bacteria: Mycobacterium tuberculosis, responsible
of the airborne and contagious pulmonary tuberculosis. The latter
is estimated by WHO as the cause in 2016 of 1.7 million deathes
over ca. 10 million new cases; immunodeficient patients (i.e. HIV-
positive patients) and emergence of strains (multi)-resistant to the
few drugs currently employed, darken the board.[12]
Problems related to infectiology and bacterial resistance are now
considered as
resistance being for the WHO one of the “biggest threats to global
a
major public health concern,[4] bacterial
[a]
[b]
Dr. M. Mourer, Pr. R. E. Duval, Pr. J.-B. Regnouf-de-Vains
Université de Lorraine, CNRS, L2CM
F-54001 Nancy, France
E-mail : maxime.mourer@univ-lorraine.fr
Dr. M. Daffé, Dr. P. Constant
With the hope to propose new molecules facing resistance (and
multiresistance) of tubercular bacilli to the active substances of
first intention (i.e. isoniazide (INH) and rifampicin) we chosen to
investigate the antimycobacterial properties of some of our
compounds. De facto, we found that our cationic derivatives
exhibited very interesting antimycobacterial activities. (i.e. <1
µgꞏmL-1 for some of them on INH-resistant MYC5165 M.
tuberculosis strain).[12-14]
Département Tuberculose & Biologie des Infections, Institut de
Pharmacologie et de Biologie Structurale, UMR 5089,
Université de Toulouse, CNRS, UPS, 31077, Toulouse Cedex 04,
France
[c]
Pr. R. E. Duval
ABC Platform®
Nancy, F-54001, France
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In the continuing search of new active compounds, we are trying Results and Discussion
to refine our data on the existence of any structure-activity
relationships. In this sense, we present here the first study
comparing the antibacterial activities of different conformers of
tetra-(guanidinopropyloxy)-tetra-tert-butylcalix[4], i.e.: cone 13,
partial cone 14, 1,2-alternate 15 and 1,3-alternate 16 presented
in scheme 1 (respectively named: cone, paco, 1,2-alt and 1,3-alt).
Compared to our previous amphiphilic compounds where the
cationic charges are tethered at the upper rim of the conic
calix[4]arene via an ethylene linker, the guanidinium subunits are
attached, in the case of the conic compound 13, at the lower rim
by means of longer propylene spacers. This difference in length
and orientation of polarity could result in modifications of activities,
but also could allow, by partial inversion of phenyl rings, the
genesis of bolaamphiphiles displaying charges on both ends of
the macrocycle. In the case of the paco 14, 1,2-alternate 15 and
1,3-alternate 16, an idealized stretched structure should approach
a length of ca. 16-19 Å, near the thickness of the membrane lipidic
bilayer (ca. 25-30 Å), prone to interactions of opposite cations with
anions of both lipid layers.
The cationic structures (especially guanidinium salts) extensively
studied in our group have shown, for most of them, very
interesting results in term of activity on a wide range of bacterial
strains. The effect of active polycationic calix[4]arene was
analyzed as the disruption of the bacterial membrane[15]
,
associated to the modification of electrophoretic mobility[15] and
increase of the membrane permeability.[16] Atomic Force
Microscopy (AFM) investigations have enabled us to verify
denaturation of the bacterial wall with loss of elasticity and holes
formation in the outer membrane.[17] Combined to
physicochemical investigations on various eukaryotic and
prokariotic membrane models with Langmuir balance,[18] these
first results have in fact validated our initial hypothesis, based on
strong synergized electrostatic interactions between constrained
positive charges of guanidinium and membrane anions
representative of the global negative charge of bacterial surface.
These results encouraged us to continue our research in this
sense, via the introduction of hydrophobic groups on the cationic
calixarenes, considering here a greater amphiphily in the aim to
generate supplementary interactions with hydrophobic membrane
constituents, in synergy with cations.
As in our previous studies, we prepared for comparative biological
studies, the formal monomeric building block of calixarenes, the
p-tert-butyl-(guanidinopropyloxy)-benzene 20 (Scheme 1).
In this study, the tetra-p-tBu-calix[4]arene associated to the
propyloxy arms will represent the lipophilic part. The cone
conformer 13 should display an amphiphilic character, resulting in
strong oriented and synergic electrostatic interactions of the
tetracationic head with the anionic components of the bacteria
surface, and orienting the additional functionalities externally or
internally, with respect to the membrane structure, for further
interactions. The declination in its various possible conformations
(i.e. partial cone 14, 1,2-alternate 15 and 1,3-alternate 16) should
allow us to control the amphiphilicity and open to bolaform
structures by modulation of the ratio cation / tert-butyl on the
calixarene rims. This should increase/strengthen the interactions
with both parts of the lipid bilayer of the bacterial membrane and
have a consequence on the antibacterial activity.
H
N
H2N
NH
O
4 CF3COOH
O
O
4 CF3COOH
O
O
O
O
O
NH
NH2
NH
NH2
HN
HN
HN
NH
H2N
HN
NH
HN
NH
HN
H2N
HN
NH
NH2
H2N
NH2
NH
14
13
CF3COOH
cone
partial cone
O
NH
NH
HN
NH
NH
HN
H2N
HN
NH2
NH2
H2N
NH
20
HN
H2N
NH
O
O
O
O
O
4 CF3COOH
O
O
O
4 CF3COOH
Synthesis
NH
NH
The general synthetic pathway leading to final guanidylated
compounds 13, 14, 15, 16 and 20 involved four steps from the
commercially available calixarene A and p-tBu-phenol C (Scheme
2). For the synthesis of this family of conformers our starting point
was based on a procedure adapted from Böhmer et al.[19]
reporting the development of various 1,3-alternate calix[4]arene
selectively functionalized by amino groups. Tetra-p-tBu-
calix[4]arene A was suspended in anhydrous THF in the
H2N
HN
H2N
NH
NH2
HN
NH
N
N
H
H
H2N
15
1,2-alternate
16
1,3-alternate
Scheme 1. Compounds of the study.
presence
of
cesium
carbonate.
After
addition
of
These five compounds were fully characterized and their in vitro
antibacterial activities i.e. MIC against Gram positive and Gram
negative reference bacteria and on M. tuberculosis, and cellular
toxicity (IC50) on non-cancerous human pulmonary embryonic
fibroblasts (MRC-5 cell line) were assessed. They show, for most
of them, very interesting antibacterial activities, combined to low-
to modest- cytotoxicity. And we demonstrated that this is directly
related to the conformation of platforms.
bromopropylphtalimide, the mixture was refluxed under Ar. After
treatment, the crude material is, according to the authors, taken
back one cycle of successive crystallization allowing each of them
the isolation of one conformer (1,3-alt, 1,2-alt and paco). A first
crystallization in a CH2Cl2-MeOH mixture, afforded white crystals
of 1,3-alternate conformer 4. If it is possible, from mother liquor,
to obtain by crystallization the two other conformers,
chromatographic purification (SiO2, CH2Cl2) was found faster and
more efficient. The three conformers 1,3-alt (4), 1,2-alt (3) and
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paco (2) were then obtained in the form of white crystalline
powder with spectral analysis consistent with the literature
data.[20] The cone conformer 1 was obtained in two steps with an
The NMR analyzes allow us to easily identify the different
conformations of the derivatives 13 to 16. For example, with 13
C
resonance signals in the range around 28-32 ppm for the C-atom
of the CH2 bridge when the attached phenolic units are in the syn-
orientation and in the range 37-40 ppm when they are in anti-
orientation.
overall yield of 60%, first by reaction of
A
with
bromopropylphtalimide and K2CO3 in refluxing MeCN to give the
diether B with a yield of 88%; B was then alkylated at the residual
OH functions by reaction in toluene with bromopropylphtalimide
using NaH as base, to give 1 with a yield of 70%. The formation
of tetraethers can be achieved in one step from the native
calixarene A with NaH in DMF [20-21], but the two steps process,
as aforementioned, appears preferable according to our
experience.
1H-NMR and 13C-NMR processed in D6-DMSO were completed
by 2D HSQC experiments in order to identify precisely the Ar-CH2-
Ar resonance signals. All NMR data enabling access to the
different conformations are shown in Table 1. The shape of the
resonance signal of the methylene bridges protons and of the
aromatic protons also confirms the conformational structure. Thus
1
the compound 13 presents a cone conformation with a H-NMR
Ar-CH2-Ar resonance signal appearing as AX system around 3.15
and 4.24 ppm (4 H for each doublet) and as a 13C-NMR singlet at
32.4 ppm. A singlet at 6.80 ppm integrating 8 protons for the
aromatic hydrogens shows a syn-orientation of all phenols, and
therefore an identical space environment for every ArH. For
compound 14, the resonance signal corresponding to the
methylene bridges is divided into an AX system at 3.04 and 3.99
ppm integrating 4 H, and a singlet at 3.64 ppm integrating 4 H,
characteristic of at least one anti-orientation of two phenolic units.
The 13C-NMR signals at 30.20 (syn-orientation) and 37.67 (anti-
orientation) confirms the partial-cone conformation. The presence
of two singlets and two doublets for the aromatic H confirms also
the inversion of one phenolic unit. Indeed, the 4 aromatic protons
of phenolic units adjacent to the reversed cycle lose their
equivalence in terms of chemical space environment. Thus there
is formation of two doublets integrating 2 H, and resulting from a
low 4J coupling (2.1 Hz). We confirm the 1,2-alternate
conformation of the derivative 15 by the presence of an AX
system (4 H) and a singlet (4 H) resonance signal for the
methylene bridges protons, and two 13C resonance signals at 28.6
and 37.8 (syn- and anti-orientation respectively). The
disappearance of the singlets of ArH to the benefit of two doublets
integrating 4 H each, confirms the reversal of two adjacent cycles.
Finally, the compound 16 exhibits a singlet resonance signal at
3.64 ppm for Ar-CH2-Ar as well as a single signal at 37.3 ppm
(anti-orientation) in 13C-NMR, characteristic of the 1,3-alternate
conformation. The aromatic H, all equivalents, appear as one
singlet at 6.99 ppm.
a
d
e
f
4
4
4
4
4
OH
O
O
O
O
O
O
CF3COOH
A
NH
O
NH2
HN
HN
N
O
O
N
N
O
NH2
c
b
H
1 (cone)
5 (cone)
9 (cone)
13 (cone)
2 (partial cone)
3 (1,2-alt)
6 (partial cone)
7 (1,2-alt)
10 (partial cone)
11 (1,2-alt)
14 (partial cone)
15 (1,2-alt)
4 (1,3-alt)
8 (1,3-alt)
12 (1,3-alt)
16 (1,3-alt)
2
OH
O
f
b
d
e
B
N
O
O
O
O
O
OH
O
C
CF3COOH
NH
N
O
O
NH
O
NH2
O
O
N
N
O
H2N
NH
H
17
18
19
20
Scheme 2. Reagents and conditions: a) Bromopropylphtalimide, Cs2CO3, THF,
Rflx, days; b) Bromopropylphtalimide, K2CO3, CH3CN, Rflx, 18h; c)
Bromopropylphtalimide, NaH, toluene/CH3CN, Rflx, 18h; d) H2N-NH2, H2O,
EtOH, Rflx, 6h; e) N1,N2-(di-Boc)-N3-triflylguanidine, CH2Cl2/MeOH, RT,
overnight; f) CH2Cl2, TFA, RT, overnight.
7
These four tetra-phtalimidopropyl conformers were then engaged
in the phtalimide cleavage process, according to a standard
procedure using hydrazine in refluxing ethanol. This afforded the
tetra-aminopropyl derivatives, cone, partial cone, 1,2-alternate
and 1,3-alternate conformers 5, 6, 7 and 8 respectively. The
guanidine functions were generated by reaction of the latter with
N1,N2-(di-Boc)-N3-triflylguanidine according to our previous works.
[8] The octa-boc derivatives 9, 10, 11 and 12 thus obtained were
finally treated with trifluoroacetic acid to give the corresponding
tetra-guanidinium salts 13, 14, 15 and 16 (cone, partial cone, 1,2-
alternate and 1,3-alternate respectively). NMR analyses (1H and
13C) and mass spectrometry were consistent with the literature for
13 [19], and with the proposed formulas for the three new salts
(Table 1). Elemental analyses were consistent with the presence
of 2 or 3 water molecules. An equivalent protocol was used to
obtain the monomeric derivative 20 from C.
NMR analysis for conformational determination
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