Synthesis of Pyridoclax Analogues: Insight into Their Druggability by Investigating Their Physicochemical Properties and Interactions with Membranes

Article abstract of DOI:10.1002/cmdc.201900542

Pyridoclax is considered a promising anticancer drug, acting as a protein-protein interaction disruptor, with potential applications in the treatment of ovarian, lung, and mesothelioma cancers. Eighteen sensibly selected structural analogues of Pyridoclax

Full text of DOI:10.1002/cmdc.201900542

Accepted Article
Title: Synthesis of new Pyridoclax analogs: Insights into their
druggability through investigation of their physicochemical
properties and their interactions with membranes
Authors: Martina De Pascale, Domenico Iacopetta, Marc Since, Sophie
Corvaisier, Veronique Vie, Gilles Paboeuf, Didier Hennequin,
Serge Perato, Marcella De Giorgi, Maria Stefania Sinicropi,
Jana Sopkova-De Oliveira Santos, Anne-Sophie Voisin-
Chiret, and Aurelie Malzert-Freon
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published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900542
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900542
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10.1002/cmdc.201900542
ChemMedChem
FULL PAPER
Synthesis of new Pyridoclax analogs: Insights into their
druggability through investigation of their physicochemical
properties and their interactions with membranes
Martina DE PASCALE,[a]* Domenico IACOPETTA,[b]* Marc SINCE,[a] Sophie CORVAISIER,[a] Véronique
VIE,[c] Gilles PABOEUF,[c] Didier HENNEQUIN,[d] Serge PERATO,[a] Marcella DE GIORGI,[a] Maria
Stefania SINICROPI,[b] Jana SOPKOVA-DE OLIVEIRA SANTOS,[a] Anne-Sophie VOISIN-CHIRET,[a]**
Aurélie MALZERT-FREON[a]**
[a]
Dr Martina DE PASCALE
Dr Marc SINCE
Miss Sophie CORVAISIER
Dr Serge PERATO
Dr Marcella DE GIORGI
Prof Jana SOPKOVA-DE OLIVEIRA SANTOS
Prof Anne-Sophie VOISIN-CHIRET, http://orcid.org/0000-0001-5564-2244
Prof Aurélie MALZERT-FREON, http://orcid.org/0000-0001-6023-1861
Normandie Univ, UniCaen, CERMN, F-14000 Caen, France
E-mail : aurelie.malzert-freon@unicaen.fr; anne-sophie.voisin@unicaen.fr
Dr Domenico IACOPETTA
Prof Maria Stefania SINICROPI
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036, Arcavacata di Rende, Italy
Dr Véronique VIE
[b]
[c]
[d]
M Gilles PABOEUF
Département Matière Molle BioMolécules aux Interfaces Fluides IPR, UR1, Campus de Beaulieu, F-35042 Rennes Cedex, France
Dr Didier HENNEQUIN
Normandie Univ, UniCaen, ABTE, F-14000 Caen, France
*Equal authorship contribution
**Corresponding Authors
Supporting information for this article is given via a link at the end of the document
Abstract: Pyridoclax is considered as a promising anticancer drug,
acting as a protein-protein interactions disruptor, with potential
applications in the treatment of ovarian, lung, and mesothelioma
cancers. 18 sensibly selected structural analogs of Pyridoclax were
synthesized, and their physico-chemical properties were
systematically assessed, and analyzed. Moreover, considering that
drug-membrane interactions play an essential role to understand
drugs mode of action and eventual toxic effects, membrane models
were used to investigate such interactions in the bulk (liposomes) and
at the air-water interface. The measured experimental data on all
original oligopyridines have permitted to assess relative differences in
terms of physico-chemical properties which could be determinant for
their druggability, and hence for their drug development.
inhibitors of Bcl-xL (Navitoclax) or Bcl-2 (Venetoclax). In this
context, great efforts have been directed towards the
development of therapeutic drugs able to inhibit Mcl-1 in cancer
cells to lead to efficient cell death.
Pyridoclax is an oligopyridine, consisting of four pyridine rings
substituted by methyl group on the ortho position of the second
ring and by a styryl group on the third ring. Using surface plasmon
resonance assay, it has been established that this lead compound
is able to bind to Mcl-1.[4] When used in combination with Bcl-xL-
targeting siRNA or with ABT-737, Pyridoclax is able to induce
apoptosis in ovarian, lung, and mesothelioma cancer cells.
Pyridoclax is not cytotoxic when it administered as a single
agent.[4] Thus, considering the dramatic situation of patients with
such tumors resistant to conventional chemotherapy, this opens
interesting perspectives concerning the use of Pyridoclax and its
derivatives in combination with Bcl-xL targeting molecules. In vivo
preclinical studies are now required to establish the potential
interest of such innovative molecule.
Introduction
The major role of Mcl-1 in the carcinogenesis of a wide variety of
tumor localizations has been established, that has gradually led
to the designation of Mcl-1 as a priority therapeutic target.[1] In
addition, the major role of the pro-apoptotic proteins Mcl-1 and
Bcl-xL overexpression and cooperation in protecting tumor cells
against apoptosis and acquiring resistance to many anticancer
drugs is now well known.[2,3] In particular, overexpression of Mcl-
1 was identified as one of the major factors of resistance to
Considering that the diverse substitutions realized on members of
the oligopyridine family, and in particular on Pyridoclax, have led
to compounds that do not scrupulously respect the 5 Lipinski’s
rules,[5,6] it appears essential to deeply characterize their physico-
chemical properties before any in vivo evaluation. Indeed, poor
solubility and/or poor permeability lead to insufficient absorption
and, in fine, to low bioavailability.[7] In these conditions, the
therapeutic potential of these molecules could be misevaluated.
1
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In the present study, we propose to experimentally determine
relevant physico-chemical properties of Pyridoclax and some of
its reasonably selected structural analogs listed in Figure 1, in
order to establish structure-physicochemical properties
relationships of these oligopyridines of therapeutic interest.
in order to investigate the influence of some structural
modifications on their physico-chemical properties. Considering
that MR29072 has a bipyridine core in the middle of the molecule,
substituted by a methyl and a styryl group in the 5 position, 1) the
influence of the number of aromatic rings, 2) the nature and
position of lateral substituents, 3) the nitrogen position on terminal
pyridines, 4) replacement of central or terminal pyridines with a
phenyl ring, and 5) functionalization by introducing polar groups
on one end were investigated in this study (Figure 1).
Results and Discussion
Library of studied Pyridoclax derivatives
Synthesis of Pyridoclax derivatives with various pyridyl-chain
length
The synthesis of oligopyridines based on Bohlmann-Rahtz
reaction highlights low yields and numerous reaction steps. To
overcome these difficulties, we have developed the Garlanding
concept which allows building a long linear chain from one ring by
the implementation of regioselective iterative cross coupling
metallocatalyzed reactions between boronic species and di- or tri-
halogenated compounds.[8–10]
This flexible and highly reproducible methodology permitted to
obtain about hundred new bi-, ter-, quater-, quinque- and sexi-
pyridines, phenylpyridines, and thienylpyridines with satisfactory
yields.[11–16] Starting from a quaterpyridine, Pyridoclax named
MR29072, we have designed and synthesized new oligopyridines
In order to study the influence of the number of aromatic rings, we
synthesized Pyridoclax analogs with 2, 3, 5 and 6 aromatic rings
as the main chain, and with or without the styryl motif. To access
the compounds MR33715 and MR33716, we used the common
synthetic intermediate 5-bromo-2-(6-iodo-5-methyl-3-pyridyl)-3-
[(E)-styryl]pyridine previously described;[4] taking advantage of
the difference in reactivity of the halogens (I>Br), the compound
1,
2-(6-bromo-3-pyridyl)-5-[5-bromo-3-[(E)-styryl]-2-pyridyl]-3-
methylpyridine, was obtained with a relative selectivity with a yield
of 72% (Scheme 1).
Figure 1. Classification of Pyridoclax derivatives from structural modifications.
2
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Scheme 1. Chemical procedure to synthesize oligopyridines MR33716, MR33715, MR29063, MR33717 and MR33714. Reagents and conditions: (a) Pd(PPh3)4 or
PdCl2(dppf) (5% mol), K3PO4 (2.5 equiv), 1,4-dioxane, reflux, 3‒24 h; (b) Pd(PPh3)4 (10% mol), base (Na2CO3 or K3PO4) (5.0 equiv), 1,4-dioxane, reflux, 2‒3 h; (c)
NaI (10.0 equiv), CH3COCl (1.5 equiv), acetonitrile, 100°C, 1 h, C = 0.4 M, microwaves.
The compound 1 was engaged either in a second Suzuki-Miyaura
cross coupling reaction with 2.2 equivalents of 3-pyridylboronic
acid to afford MR33716 (57% yields) or, in a halogen-halogen
exchange to exploit the difference of the reactivity between iodine
and bromine. The bromo-iodo-compound 2 was then engaged in
two successive Suzuki-Miyaura cross coupling reactions with 6-
bromo-3-pyridylboronic acid and 3-pyridylboronic acid to give
MR33715 (51% yields). MR29063 has been produced (61%
yields) by the implementation of a double Suzuki-Miyaura cross
coupling reaction from (E)-5-bromo-2-chloro-3-styrylpyridine,[4]
with 2.5 equivalents of 3-pyridylboronic acid: in this case, the
nature and the position of halogens on the pyridine ring do not
allow a regioselective coupling reaction, making it possible to
obtain the expected terpyridine.
To obtain MR29015 (Scheme 2), 5-bromo-2-iodo-3-
methylpyridine was engaged in a Suzuki-Miyaura cross coupling
reaction with 1.25 equivalents of 6-bromo-5-methyl-3-
pyridylboronic acid to give 2-bromo-5-(5-bromo-3-methyl-2-
pyridyl)-3-methylpyridine 5, which is leading to 5-bromo-2-(6-
iodo-5-methyl-3-pyridyl)-3-methyl-pyridine
6 via a halogen-
halogen exchange. The bromo-iodo-pyridine 6 was then engaged
in a second Suzuki-Miyaura cross coupling reaction with 1.25
equivalents of 6-bromo-5-methyl-3-pyridylboronic acid to give 5-
bromo-2-[6-(6-bromo-5-methyl-3-pyridyl)-5-methyl-3-pyridyl]-3-
methyl-pyridine 7. The last step consists in a third Suzuki-Miyaura
cross coupling reaction with 2.5 equivalents of 3-pyridylboronic
acid to give MR29015 (74% yields).
MR29004 and MR29016, respectively four and six pyridine ring
compounds, were synthesized using Garlanding methodology as
it was previously described.[17]
From 2-chloro-3-iodopyridine, we obtained MR33717 (by the
implementation of an iterative Suzuki-Miyaura cross coupling
reaction with, firstly (E)-styrylboronic acid to obtain intermediately
(E)-2-chloro-3-styrylpyridine 4 (52% yields) and, secondly with 5-
methylpyridin-3-ylboronic acid to produce MR33717 (65% yields).
3
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Synthesis of Pyridoclax derivatives with substituted phenyl rings
To prepare the bisphenylpyridine MR30821, we used the strategy
described in Scheme 3. In one hand, we first synthesized
phenylpyridine 8 from 3-pyridylboronic acid and 2-bromo-5-
iodotoluene with a yield of 85%. The use of a non-halogenated
boronic acid limits side reactions and confirms the difference in
reactivity between the iodine and bromine atoms, despite the
presence of a bulky methyl substituent. Phenylpyridine 8 was then
engaged in a halogen-metal exchange reaction carried out using
n-butyllithium in THF at -78°C, followed by the addition of
triisopropyl borate to easily conduct to the corresponding boronic
acid
9 with 68% yield. In the other hand, from 2,5-
dibromobenzaldehyde, the aldehyde 10 is obtained with a good
yield of 80% and selectively without trace of its isomer because of
the presence of the aldehyde function which makes possible the
orientation of the cross coupling reaction towards the formation of
the carbon-carbon bond in ortho position. Then, the aldehyde 10
was engaged with boronic acid 9 in a Suzuki-Miyaura cross
coupling reaction leading to the formation of bisphenylpyridine 11
Scheme 2. Chemical procedure to synthesize oligopyridine MR29015.
Reagents and conditions: (a) Pd(PPh3)4 (5% mol), Na2CO3 (2.5 equiv), 1,4-
dioxane, reflux, 20 h; (b) NIS (1.1 equiv), acetonitrile, reflux, 5 h; (c) Pd(PPh3)4
(10% mol), Na2CO3 (5.0 equiv), 1,4-dioxane, reflux, 20 h.
with excellent yield (93%).
A Horner-Wadsworth-Emmons
Synthesis of Pyridoclax derivatives with a carboxylic acid function
on one end
reaction was carried out in the presence of 1 equivalent of
phosphonate and 2 equivalents of potassium hydroxide and leads
to styrylphenylpyridine MR30821 with good yields (85%).
A relatively similar procedure, previously described,[4] was used
to access to MR30820.
The implementation of iterative cross-coupling reactions between
boronic species and di- or tri-halogenated compounds whose
reactivity is different because of their nature or their position on
the pyridine, allowed us to obtain Pyridoclax derivatives as (E)-2-
(3-(5-methyl-3'-styryl-[3,2':5',3''-terpyridin]-6-yl)phenyl)acetic acid
MR31366 and (E)-2-(3-(5'-methyl-3-styryl-[2,3':6',3''-terpyridin]-5-
yl)phenyl)acetic acid MR31386.[18]
Scheme 3. Chemical procedure to synthesize oligopyridine MR30821. Reagents and conditions: (a) Pd(PPh3)4 (5% mol), Na2CO3 (2.5 equiv), 1,4-dioxane, reflux,
20 h; (b) n-BuLi (1.3 equiv), triisopropylborate (1.3 equiv), THF dry, -78 °C, 2 h; (c) KOH (2.0 equiv), THF, reflux, 20 h.
Synthesis of Pyridoclax derivatives without pyridines at ends or
with a different nitrogen position on terminal pyridines
Concerning the preparation of Pyridoclax analogs MR30814,
MR31348, MR31349 and MR31350, either they were synthesized
using 2.5 equivalents of corresponding (het)aryl boronic species
in order to produce analogs with identical ends (MR30814 and
MR31350), either, they were prepared with two different (het)aryl
boronic species in order to produce analogs with different ends
4
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10.1002/cmdc.201900542
ChemMedChem
FULL PAPER
(MR31348 and MR31349). Moreover, the choice of the synthesis
pathway was determined in function of the reactivity of different
boronic species as it was previously described.[4]
Then, the synthesis of boronic ester of bromobipyridine and its
cross coupling reaction with bromobipyridine 12 was realized in
an one-pot reaction.The quaterpyridine MR33718 was obtained
with good yield (56%).
Synthesis of Pyridoclax derivatives with various lateral alkyl
substituents
All 19 studied compounds were synthesized (procedures are
detailed in the Experimental Section) and each structure was
characterized by standard methods (1H- and 13C-NMR).
MR29005 was synthesized using Garlanding methodology as it
was previously described.[17] Concerning the symmetric
quaterpyridine MR33718, 2,5-dibromopyridine was engaged in a
Suzuki cross coupling reaction with 3-pyridylboronic acid to give
5-bromo-2-(3-pyridyl)pyridine 12 (Scheme 4).
Determination of the fundamental physico-chemical
properties of Pyridoclax and of its derivatives
To predict successful drug development from molecular
properties, Lipinski proposed “the rules of 5” (RO5) relating to the
importance of molecular weight (< 500 g/mol), the number of
solvent hydrogen-bond donors (HBD) and acceptors (HBA) (< 5
HBD, < 10 HBA), the logP value (calc logP < 5).[5] In addition to
these RO5, other properties have also to be assessed as they are
considered to influence oral bioavailability.[19] So, for each
compound, calculated logP (i.e., calc logP), acidity (i.e., calc pKa),
HBA ability, molecular weight, the number of rotatable bonds, that
of aromatic rings into the scaffold, the number of lateral alkyl
substituents, and the polar surface area (i.e., PSA) were
estimated using computational chemistry programs. Each
compound was also evaluated experimentally for lipophilicity
(chrom logP), thermodynamic solubility, and effective permeation
rate (i.e., Pe) (Table 1).
Scheme 4. Chemical procedure to synthesize oligopyridine MR33718.
Reagents and conditions: (a) Pd(PPh3)4 (5% mol), Na2CO3 (2.5 equiv), 1,4-
dioxane, reflux, 20 h; (b) bispinacolato diboron (0.52 equiv), PdCl2 (dppf) (10%
mol), KOAc (2.5 equiv), 1,4-dioxane, reflux, 3 h.
pKa
As all of the studied compounds present at least two pyridines in
their scaffold, the acidic dissociation constants were calculated in
order to explore ionization state of the studied compounds.
Table 1. Calculated and experimental physico-chemical properties of the studied oligopyridines. Molecular weight (MW), acidity (i.e., pKa), n-octanol/water partition
coefficient (i.e., calc logP), the number of hydrogen-bond acceptors (i.e., ♯ HBA), of rotatable bonds, of aromatic rings in scaffold, of lateral alkyl substituents, and
the polar surface area (i.e., PSA) were calculated using ChemAxon software; the lipophilicity was also experimentally determined by chromatographic method (i.e.,
chrom logP); thermodynamic solubility in aqueous buffer (i.e., aq. solub.) was determined by UHPLC; the effective permeation rate (Pe) was determined by PAMPA
assay; n.d. means not determined.
Cpd
[MR]
MW
[g/mol]
♯ aromatic
rings in
scaffold
♯ lateral alkyls
substituents
♯ HBA
♯ Rotatable
bond
PSA
[Å2]
Calc
pKa
Calc
logP
Chrom
logP
Aq. Solub.
[µM]
Pe
[nm/s]
29072
33717
29063
33716
33715
33714
29004
29015
29016
33718
29005
30814
31348
31349
31350
30820
30821
31386
31366
426.5
272.3
335.4
503.6
580.7
247.1
338.1
429.2
520.0
310.1
324.1
424.5
425.5
425.5
426.5
424.5
424.5
483.5
483.5
4
2
3
5
6
3
4
5
6
4
4
4
4
4
4
4
4
4
4
1
1
0
1
1
1
2
3
4
0
1
1
1
1
1
1
1
1
1
4
2
3
5
6
3
4
5
6
4
4
2
3
3
4
2
2
5
5
5
3
4
6
7
2
3
4
5
3
3
5
5
5
5
5
5
7
7
45.0
22.5
33.8
56.3
67.6
33.8
45.0
56.3
67.6
45.0
45.0
22.5
33.8
33.8
45.0
22.5
22.5
71.9
71.9
4.84
4.81
4.80
4.85
4.86
4.85
4.89
4.91
4.94
4.66
4.87
4.17
4.43
4.75
4.92
5.01
5.01
4.21
4.47
5.83
4.69
4.59
6.6
4.4
3.7
3.7
4.6
5.3
1.7
2.5
3.1
3.7
2.6
2.5
6.8
5.9
5.7
4.4
6.4
6.3
ND
ND
9.4 ± 0.1
138.1 ± 6.4
18.9 ±0.4
5.8 ± 2.1
n.d.
160
891
1,200
21
7.37
2.35
3.58
4.82
6.06
2.65
3.12
8.45
7.14
7.14
5.83
7.66
7.66
6.77
6.77
n.d.
1,130
260
0
384 ± 43
26.8 ± 3.5
n.d.
n.d.
n.d.
719
1,200
0
n.d.
70.9 ± 3.1
0.7 ± 0.8
2.5 ± 2.9
1.2 ± 0.6
20.5 ± 0.7
1.1 ± 0.4
< 1
150
160
160
2
0
>250
470
270
> 250
5
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pKa values were estimated at physiological pH of 7.4, using
ChemAxon software (Marvin 16.1.4.0, 2016, ChemAxon
(http://www.chemaxon.com/)), with a method based on the partial
charge calculation of atoms in the molecule. Only the most basic
pKa was retained (Table 1). Calc pKa values appear as very
narrowly distributed, and remain lower than 5.1. Except MR31366
and MR31386, that have a meta-phenylacetic acid group at one
of terminal pyridines (acidic pKa of 5.01, and 4.78, respectively),
all synthesized compounds present a predominant unionized form
at pH 7.4. It should be pointed out that molecules with pKa values
lower than 8, as in the present case, are expected to avoid
interactions with P-glycoprotein (Pgp),[20,21] and thus, should
bypass unfavorable mechanisms as multi-drug resistance.
values range from 1.7 to 3.7. For the series characterized by the
presence of a lateral hydrophobic styryl group (MR33717,
MR29063, MR29072, MR33716, and MR33715), chrom logP
values are comprised between 3.7 to 5.3. In both series, addition
of pyridine into the garland framework leads to a proportional
increase of the chrom logP value of about 0.6 per added unit
(regression coefficient R2 > 0.94).
Solubility
Thermodynamic solubility was measured using a miniaturized
shake-flask method for all compounds at the exception of
MR33718, MR33715, MR29015, and MR29016 whose solubility
in DMSO or in water/acetonitrile mixtures was too low to produce
standard solutions. In particular, for MR33715, and MR29016,
insolubility can be related to the high number of aromatic rings in
the garland scaffold (>5), and the resulting high molecular weight
(>500 g/mol). Indeed, the molecular weight of compounds
appears as negatively impact their solubility. On the contrary,
solubility seems being positively influenced by PSA and the
number of rotatable bonds. Although a moderate chrom logP
value (2.6) and a low molecular weight (310.1 g/mol), MR33718
appears totally insoluble in water and in DMSO. Such a result
could be related to the fact that MR33718 is a planar and a
symmetric compound, which can hamper its solubilization.[29] The
fact that this compound has a negligible dipole moment value
(0.001 Debyes as determined using Discovery Studio software)
and a high melting temperature (198°C) corroborates this idea.
The solubility of MR31366 and MR31386, ionized compounds,
was found higher than 250 µM, i.e. the concentration used to
prepare supersaturated solutions in our experimental conditions
(see Experimental Section).
Lipophilicity
Lipophilicity is a major physico-chemical parameter to consider in
drug design because of its omnipotent impact on druggability
parameters (i.e. solubility and permeation),[22,23] as well as on in
vivo pharmacokinetic parameters (i.e. absorption, plasma protein
binding, phase
I
metabolization, and hERG binding).[5,24]
Logarithms of partition coefficients (logP) were first calculated
using several software programs, i.e., Discovery Studio,
Schrödinger, ChemAxon, and Molinspiration. In parallel, except
for ionized MR31366 and MR31386 at pH=11.5, the logP (chrom
logP) were also measured using an isocratic liquid
chromatography method, in non-ionized conditions.[25,26] The best
correlation (R=0.9101) between calc logP and chrom logP values
was found using the ChemAxon method based on the atomic logP
increments.[27] Considering that the calculated values were
systematically higher than those measured, the softwares should
present some limitations to efficiently predict partition coefficient
values in these oligopyridines. Such results highlight the need of
an experimental determination as suggested in other studies.[28]
In regard of the chrom logP values varying from 1.7 to 6.8 (Table
1), the studied compounds can be considered as moderately to
highly lipophilic.
Other studied molecules show a low aqueous solubility, ranging
from 0.7 to 384 µM. Such a result is reliable to the absence of
solvent hydrogen-bond donors (HBD), their lipophilicity, and
hence, of the number of the garland units and of the nature of the
lateral substituents as previously discussed. Moreover, by
MR29072, MR30814, MR31348, MR31349, MR31350, MR30820, comparing MR29072 and MR31350, it appears that the position
and MR30821 are close molecular weights compounds
constituted by 4 aromatic rings in garland, and with a methyl and
a styryl group on the central rings. In this series, substitution of a
pyridine by a phenyl ring leads to an obvious and expected
increase in chrom logP values, as observed by comparing
MR29072, MR31350 (4 Pyr, chrom logP = 4.4) to MR31348,
MR31349 (1 Phe, 3 Pyr, chrom logP = 5.9 and 5.7, respectively),
and to MR30821, MR20820, MR30814 (2 Phe, 2 Pyr, chrom logP
= 6.3 to 6.8). If the phenyl groups are positioned in the middle or
at the ends of the molecule, chrom logP range in the same order
of values (MR30814, MR30821, MR30820). Thus, the presence
of a nitrogen atom directly influences the lipophilicity of the studied
compounds: it may explain the relative polarity and the weak
contribution in global molecular lipophilicity.
The influence of the nature of the lateral alkyl substituents on the
lipophilicity of oligopyridines was also studied. By considering the
quaterpyridines MR33718, MR29005, and MR29004, it appears
that the substitution by methyl groups on the two central rings
doesn’t significantly impact chrom logP.
On the contrary, replacement of a methyl substituent by a styryl
group leads to higher molecular weight compounds, with
significantly higher chrom logP values. Indeed, for MR33714,
MR29004, MR29015, MR29016, a series of oligopyridines with an
increasing number of 3-methylpyridinyl groups, chrom logP
of the nitrogen groups on terminal pyridines could slightly
influence their solubility.
Aqueous solubility is essential for drug candidates.[30] As poor
aqueous solubility is likely to result in poor absorption, various
solubilization approaches should be further studied, as chemical
strategies based on salt formation and prodrug synthesis, or
advanced formulation strategies.[31]
Permeation ability
Using a parallel artificial membrane permeability assay (PAMPA),
a high throughout technique commonly used in drug discovery to
predict passive permeation through biological membranes,[32] the
ability of the synthesized garland compounds to cross the
gastrointestinal tract (GIT) was evaluated. Because of their
complete insolubility in water, even in presence of DMSO,
PAMPA studies could not be performed on MR33715 and on
MR29016, two compounds with 6 aromatic rings.
A threshold value of Pe > 100 nm/s is the generally accepted
threshold value to consider that permeation is not a limiting factor
for oral bioavailability.[19] At the exception of MR33716, MR29015,
MR30814, MR30820, MR30821, all synthesized molecules
present acceptable Pe values (Table 1).
According to the Lipinski’s rules,[5] to favor permeation of
compounds, the sum of H-bond donors and acceptors via -NH, -
6
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10.1002/cmdc.201900542
ChemMedChem
FULL PAPER
OH, -N and -O atoms must be lower than 5 and 10, respectively.
Furthermore, Veber et al. demonstrated dependencies of Pe on
the polar surface area (PSA) and on the flexibility of the molecule
quantified from the number of rotatable bonds (RB).[19] They
established selective criteria as follows: PSA ≤ 140 Ų, and RB ≤
10. In our molecule pipeline, despite a wide range of Pe (0 to
1,200 nm/s), no compound possesses HBD while HBA, RB, and
PSA values remain below fixed limits.
Unilamellar
phosphatidylcholine (PC),
Vesicles
(LUVs)
based
on
soybean
a
mixture of phospholipids with
different fatty acids (linoleic acid in majority) mimicking the natural
membranes,[39] are widely used as a classical lipid bilayer
membrane model.[40] 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-
glycerol (DOPG) was added in a w/w ratio of 4:1 to give 20 %
negatively charged head groups, within typical physiological
range.[41] Since cholesterol is a constituent present in most
mammalian cell membranes, and it is known for its effects on
membrane stability and fluidity, liposomes were also formulated
with defined proportions of cholesterol. The oligopyridines were
incubated in presence of LUVs (see supporting information for
their granulometric properties, Table S1), and their emission
fluorescence spectra were recorded (see Figure 2 for MR29072,
and the supporting information part for other compounds, Figure
S1).
Hydrophobicity is often considered as a key physicochemical
property that contributes to the passive membrane permeation of
a compound by governing the membrane partition process.[22]
Often, for moderately lipophilic compounds, a positive relationship
exists between Pe and calc logP while for high lipophilic
derivatives (calc logP > 3.5), the higher the logP, the worst the
permeation rate is.[33] In our case, no permeable MR30814,
MR30820, and MR30821 have high chrom logP (≥ 6.3). Presence
of 2 phenyl groups in these molecules renders them very poorly
soluble and no permeable. So, such compounds should be
discarded since they will not permit acceptable oral bioavailability.
Moreover, considering the absorbance spectra obtained during
PAMPA experiments (absorbance levels are lower at the end than
at the beginning, results not shown), these compounds may be
retained within the membrane because of their high lipophilicity.
Permeation within the membrane imposes a decrease in entropy
on the molecule whose the free energy cost depends on the size
of the permeant.[34] Pe is negatively correlated to the molecular
weight. Considering the no permeable MR33716 and MR29015,
formed of 5 aromatic rings, it appears that the number of garland
units should be fixed to 4 to permit passive diffusion through
membranes, whatever the nature of the lateral alkyl substituents.
In these conditions, the molecular weight of compounds remains
lower than 500 g/mol as fixed in RO5.[5]
Figure 2. Emission fluorescence spectra of MR29072 with
recorded at concentrations, in absence or in presence of biomimetic
membranes (LUVs formulated A. without (PC:DOPG LUVs) or B. with
cholesterol PC:DOPG:chol LUVs): a. MR29072 at 10 µM; b. MR29072
MR29072 at 10 µM + LUVs; d. MR29072 at 25 µM +
LUVs.
exc=285 nm,
2
at 25 µM; c.
LUVs; e.
In regard of these different results, the lead of this family,
MR29072 presents acceptable druggability parameters, reliable
to a maximum of 4 aromatic rings, and a styryl group on a pyridine.
The presence of a carboxylic function at one end (whatever its
position) can highly help to enhance molecules hydrophilicity, and
solubility in water.
However, such foldamers with more balanced physicochemical
properties must also have the potential to be suitable chemical
biology tools.
In absence of LUVs, MR29072 spontaneously fluoresces, all the
more than its concentration is high (Figure 2). This is also the case
for the other molecules substituted by a styryl group in the ortho
position of the central junction. Such a fluorescence capacity can
be valorized, in particular to experimentally assess its partition
coefficient, by requiring only few drug amounts, by raw
fluorescence spectrophotometry in a microplate assay.[40]
In presence of PC:DOPG LUVs, a blueshift and an increase of
fluorescence intensity are observed, reflecting the transition of
MR29072 from a polar (water) to a less polar environment
(membrane), and the immobilization of the compound within the
membrane (Figure 2). Such changes in fluorescence spectra are
also present for all compounds substituted by a styryl group,
except for MR31350 for which only the increase of fluorescence
is detected. They are less objectified with LUVs formulated with
cholesterol.
For compounds having more than 4 units in their scaffold
(MR33716) or being substituted at the end by a polar group
(MR31366, MR31386), neither shift nor fluorescence increase are
observed. The presence of the LUVs did not affect the
fluorescence of compounds without styryl. If incubation of
oligopyridines is made with PC:DOPG:chol LUVs, fluorescence
intensity does not vary anymore, except for MR30821.
Determination of drug-membrane interactions
In a medicinal chemical perspective, it may be important to
evaluate drug-membrane interactions. Indeed, the chemical
compounds penetration and/or their location within biological
membranes may result in alterations of membrane permeability,
or changes in the performance of the cell.[35] In parallel, drug-
membrane interactions can contribute to explain the molecular-
level mechanisms of action of the drugs and even sometimes the
side-effects. Considering that studies performed on anticancer
drugs have established a correlation between their cytotoxic
activity, their location within membrane,[36] and their effect on
membrane stability or fluidity,[37] interactions between the
oligopyridines and biomimetic membranes have been
investigated.
Obtained experimental results suggest possible interactions
between some of the studied oligopyridines and LUVs.
To further examine these effects on membrane permeability,
Study of the interactions of the oligopyridines with liposomes as
membrane models
Liposomes were used as membrane models since they show
many physical features very close to natural membranes.[38] Large
LUVs
containing
a
self-quenching
concentration
of
carboxyfluorescein (CF), and formulated without or with
cholesterol, were exposed to each oligopyridine (10 or 25 µM).
7
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10.1002/cmdc.201900542
ChemMedChem
FULL PAPER
Any change in membrane permeability causes the self-quenched
CF to leak out the LUVs into the external medium where the probe
becomes highly fluorescent.[42]
Moreover, the hydrophilicity of both molecules probably leads to
lowering of the hydrophobic driving force for the binding.[45]
The presence of cholesterol in the phospholipid membrane
bilayer largely influences the levels of quenching of CF by
oligopyridines. Even at the highest tested concentration, only two
molecules are able to induce CF efflux: MR30820 and MR30821,
two highly hydrophobic compounds.
Cholesterol greatly influences the depth of molecules in
membranes[46]. Since after intercalation between the fatty acid
chains of phospholipids,[47] cholesterol presents a membrane
condensing effect[48], it is coherent that interactions between
oligomers and bilayers decrease in case of PC:DOPG:chol LUVs.
Potential interactions between oligomers and biomimetic
membranes may be of interest for anticancer molecules. For
example, for compounds like tamoxifen, a drug widely used in the
treatment breast cancer and also brain, liver and pancreas tumors,
it was shown that the perturbation of the lipid bilayer structure by
this amphiphilic molecule of highly lipophilic character may
contribute to its multiple mechanisms of anticancer action.[37]
Therefore, such effects should be taken into consideration to
interpret results of the pre-clinical assays performed on the
studied oligopyridines, and in particular those with a styryle, in the
future.
100 % CF efflux quasi-systematically occurs from PC:DOPG
LUVs for the compounds with a styryl group, used at 25 µM (Table
2). Except MR33715 whose effects could not be assessed
because of its lack of solubility, only the low permeable MR33716
does not lead to CF efflux. For MR31350, for which effect is not
obvious on PC:DOPG LUVs, since the blue shift is not observed,
only 55% CF release was quantified. In all cases, the
granulometric properties of the LUVs (monodisperse mean
diameter of about 100 nm) remained unchanged, as determined
by Diffusion Light Scattering (DLS) measurements at the end of
the experiments (results not shown), even if permeabilization
effects are observed.
Table 2. Study of interactions between compounds (10 or 25 µM) and
biomimetic membranes quantified from percentage of carboxyfluorescein (CF)
release (λex = 485/20 nm / λem = 528/20 nm) from large unilamellar liposomes
(LUVs), formulated with a mixture of phosphatidylcholine (PC) and 1,2-dioleoyl-
sn-glycero-3-phospho-rac-1-glycerol (DOPG) without or with cholesterol (chol),
after 4h of incubation with studied oligopyridines at room temperature.
% CF release from PC:DOPG LUVs % CF release from PC:DOPG:chol LUVs
The membrane location of oligopyridines was further studied by
fluorescence quenching measurements using DPH and TMA-
DPH as fluorescence probes. The precise positions of these
fluorophores along the membrane depth plane are well
established: DPH probe is supposed to have a deep location and
a parallel alignment to the acyl chains while TMA-DPH probe, due
to its amphipathic character, is anchored to the surface of the
membrane, within the phospholipids polar head groups.[37,49] The
Cpd [MR]
10 µM
25 µM
10 µM
25 µM
Influence of the number of aromatic rings in oligopyridines with a styryl group
33717
29063
29072
33716
33715
78
45
100
2
100
100
100
8
20
4
35
0
67
30
61
0
n.d.
n.d.
n.d.
n.d.
Influence of the number of aromatic rings in oligopyridines without a styryl group but with
methyl group(s)
33714
29004
29015
29016
0
5
0
5
0
8
6
0
0
0
0
0
2
2
extent of fluorescence quenching of
a membrane bound
fluorophore provides a measure of the accessibility of the
quencher (here, the studied oligopyridine) to the fluorophore.
Consequently, it is also possible to estimate the quencher
molecule’s location within the lipid bilayer.[50] Fluorescence
quenching can be described by the Stern-Volmer equation[51]
I0/I = 1 + KSV [Q] [1]
22
10
Influence of the nature of the lateral alkyl substituents
33718
29005
29004
29072
0
1
5
3
7
8
0
0
0
0
3
2
:
100
100
35
61
where I0 and I are the corrected fluorescence intensity of the
fluorophores (DPH or TMA-DPH) in the absence and presence of
the drug, respectively; [Q] is the concentration of the quencher
(oligopyridine); KSV is the Stern-Volmer constant.
The sensitivity of DPH or TMA-DPH to the fluorescence
quenching by the oligopyridines is rather weak in comparison with
our reference, rifampicin.[52] From results (Table 3), only
compounds having 4 rings, either phenyls and/or pyridines, with
a styryl group attached on a central pyridine are able to quench
the probes when they are used at the concentration of 25 µM. In
particular, MR31348 and MR31349, which were found permeable
but highly hydrophobic (chrom logP > 5) would be more
particularly able to accumulate in the acyl chain regions.
Influence of the presence of pyridines at ends / of nitrogen position on terminal pyridines
30814
31348
31349
29072
31350
26
84
92
100
15
100
100
100
100
55
7
8
15
35
1
33
78
64
61
15
Influence of the substitution of pyridines by phenyl rings
29072
30814
30820
30821
100
26
100
72
100
100
100
100
35
7
37
30
61
33
100
100
Influence of the presence of a carboxylic acid function at one end
29072
31386
31366
100
3
1
100
4
1
35
0
0
61
3
0
In some cases, the interactions between molecules and
membranes, manifested as changes in the physical and
thermodynamic properties of the bilayers, are accompanied by
undesirable side effects.[53] Thus, drug-membrane interaction
studies may constitute a preliminary step to the biological
evaluation of their toxicity.[52]
The presence of the bulky hydrophobic group (styryl) facilitates
binding to liposomes through hydrophobic interactions, as
reported for other amphipathic -helices.[43,44]
No CF release was observed for MR31386 and MR31366. In this
case, thanks to the presence of the carboxylic acid moiety, ionized
molecules might establish repulsive electrostatic interactions with
the anionic head groups of DOPG moieties of the membranes.
Thus, in spite of the styryl, interactions between these
oligopyridines and negatively charged lipid bilayers cannot occur.
8
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10.1002/cmdc.201900542
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FULL PAPER
proteins are capable of oligomerize as a result of apoptotic stimuli
to form pores in the mitochondrial outer membrane, thus allowing
the release of cytochrome c into the cytoplasm. In turn,
cytochrome c induces the activation of caspases leading to
apoptosis.[54] Taking into account that pro-apoptotic members of
the Bcl-2 protein family induce increase of outer membrane
permeability, while anti-apoptotic members prevent this, it seems
important to consider with attention the ability of the oligopyridines
to interact with membranes.
Table 3. Study of membrane location of oligogypridines from quantification of
fluorescence quenching (I0/I as described by the Stern-Volmer equation) of the
probes DPH and TMA-DPH in LUVs, formulated with
a mixture of
phosphatidylcholine (PC) and 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol
(DOPG) without or with cholesterol (chol), after 4h of incubation with studied
compounds at room temperature.
I0/I of DPH
in PC:DOPG PC:DOPG:chol in PC:DOPG in PC:DOPG:chol
LUVs LUVs LUVs LUVs
I0/I of DPH in I0/I of TMA-DPH I0/I of TMA-DPH
Cpd [MR] 10 µM 25 µM 10 µM 25 µM 10 µM 25 µM 10 µM 25 µM
Figure 3 summarizes the highlighted interactions between the
structural modifications performed on Pyridoclax derivatives and
their resulting physico-chemical properties. This summary was
reinforced by a multivariate statistical treatment by Principal
Component Analysis, and a PLS regression analysis performed
on parameters that could be encrypted (see supporting
information, Figures S2-S5).
Influence of the number of aromatic rings in oligopyridines with a styryl group
33717
29063
29072
33716
33715
1.1
1.0
1.3
1.0
n.d.
1.1
1.1
1.2
0.8
n.d.
0.9
1.0
1.3
1.1
n.d.
0.8
1.1
1.4
1.2
n.d.
1.1
1.0
1.2
1.3
n.d.
1.2
1.1
1.2
1.4
n.d.
1.0
1.0
1.1
1.4
n.d.
1.1
1.0
1.0
1.1
n.d.
Influence of the number of aromatic rings in oligopyridines without a styryl group but with
methyl group(s)
The concomitant presence of a styryl group on a pyridine with 4
aromatic rings appears as a maximum to confer good druggability
parameters to the studied oligopyridines. This is exactly the case
of the lead of this family, MR29072. Interactions between the
styryle and biological membranes could occur, which is important
to take into consideration during their biological evaluation.
The chemical modifications made on the Pyridoclax derivatives
will necessarily impact their pro-apoptotic activity. In other studies,
we had previously evaluated the ability of some oligopyridine
analogs to sensitize IGROV1-R10 ovarian cancer cells to siRNA-
mediated Bcl-xL silencing, and hence, to induce apoptosis through
Mcl-1 inhibition.[4,18] From the obtained results, it appeared that
some modifications did not affect the activity, whereas others
decreased or completely abolished it in comparison with
Pyridoclax. In particular, the methyl and styryl hydrophobic motifs
situated on the bipyridine core are necessary for the pro-apoptotic
activity, as the the bipyridine core itself; the nitrogen present on
terminal pyridines also plays an important role whereas the
absence of nitrogen on one of the terminal rings does not
influence its pro-apoptotic activity. Interestingly, these structure-
activity relationships have been added to the summary proposed
Figure 3.
33714
29004
29015
29016
1.0
1.0
1.2
1.0
0.9
1.0
1.1
1.1
0.9
1.0
1.0
1.0
1.1
1.0
1.0
1.1
1.0
1.0
1.1
1.1
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Influence of the nature of the lateral alkyl substituents
33718
29005
29004
29072
1.0
1.0
1.0
1.3
0.9
1.0
1.0
1.2
0.8
0.9
1.0
1.3
1.0
1.0
1.0
1.4
1.0
1.0
1.0
1.2
1.0
1.1
1.1
1.2
1.0
1.0
1.0
1.1
1.1
1.0
1.0
1.0
Influence of the presence of pyridines at ends / of nitrogen position on terminal pyridines
30814
31348
31349
29072
31350
1.1
1.2
1.1
1.3
1.4
1.2
1.6
1.4
1.2
1.4
1.1
1.3
1.2
1.3
1.2
1.2
1.5
1.6
1.4
1.3
1.1
1.2
1.1
1.2
1.1
1.5
2.0
1.5
1.2
1.2
1.2
1.2
1.2
1.1
1.0
1.3
1.5
1.3
1.0
0.9
Influence of the substitution of pyridines by phenyl rings
29072
30814
30820
30821
1.3
1.1
1.0
1.0
1.2
1.2
1.0
0.9
1.3
1.1
1.1
1.0
1.4
1.2
1.1
1.0
1.2
1.1
1.0
1.0
1.2
1.5
1.0
1.0
1.1
1.2
1.0
1.0
1.0
1.3
0.8
0.8
Influence of the presence of a carboxylic acid function at one end
29072
31386
31366
1.3
1.1
1.1
1.2
1.2
1.2
1.3
1.1
1.2
1.4
1.3
1.3
1.2
1.0
1.3
1.2
1.3
1.7
1.1
1.1
1.1
1.0
1.1
1.0
The present oligopyridines have been conceived as Mcl-1
inhibitors.[4] Mcl-1 blocks the progression of apoptosis by binding
and sequestering the pro-apoptotic proteins Bak and Bax. These
Figure 3. Summary of the impact of the structural
modifications made on the studied derivatives on their
physico-chemical properties in comparison with
Pyridoclax (MR29072):
= means “no significant
influence”,  means “increase” and , “decrease
9
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FULL PAPER
At the present, MR29072, Pyridoclax, is considered as the lead
compound of the family of oligopyridines that we studied. In its
envisaged therapeutic application, Pyridoclax must inhibit protein-
protein interactions between Bcl-2 proteins control of the
Mitochondrial Outer Membrane Permeabilization (MOMP), which
leads to downstream caspase to execute apoptosis. Considering
this point and our results obtained so far, we have decided to
further investigate the membrane insertion property of Pyridoclax.
From ellipsometry measurements, the interfacial monomolecular
film seems to reach equilibrium, whereas the increasing surface
pressure in time suggests lateral aggregation of the compound at
the interface.
From results (Figure 5), at 10 µM, MR29072 is able to interact
within the phospholipid monolayer at a surface pressure (Π)
around 30 mN/m. Indeed, in presence of MR29072 in the
subphase, the ellipsometric angle was constant for 100 min and
then was slowly decreasing while the surface pressure decrease
began earlier at 50 min. These physical parameters were stable
for the lipid film without Pyridoclax for 6 hours (data not shown).
By binding to polar heads, Pyridoclax would create a condensing
effect of the DOPC:DOPG monolayer.
Interactions between Pyridoclax and lipid monolayer studied at
the air-water interface
Results of ellipsometry and surface pressure measurements
performed on Pyridoclax (MR29072) used at the concentration of
10 µM show that the compound is able to adsorb at the air-HEPES
interface, indicating an amphipathic behavior reliable to its
chemical structure (Figure 4).
Figure 6. Atomic Force Microscopy (AFM) images of the interfacial films
obtained at the end of kinetic interaction between MR29072 (A. 10 or B. 25 µM)
and DOPC:DOPG film. The z scale corresponding to the color scale is in the
range of -10 to 10 nm for A. and of -50 to 50 nm for B. The image size is 40 µm
x 40 µm.
From observations performed by Atomic Force Microscopy (AFM)
after transfer of the interfacial film to freshly cleaved mica sheet
(Figure 6.A.), it appears that this interaction is accompanied by
the formation of patches regularly dispersed on the images which
have a mean height of 2.5±0.2 nm and a diameter varying
between 200 to 800 nm. The presence of these domains is only
observed for the experiment made in presence of the Pyridoclax,
revealing the presence of the Pyridoclax on the lipid film. The
height of each domain is regular, meaning lateral interactions in
the film thickness avoiding 3D aggregation.
Figure 4. Ellipsometry (+) and surface pressure (o) measurements performed
on MR29072 (10 µM) at the air-buffer (HEPES 50 mM + NaCl 107 mM) interface.
Increase of the concentration of Pyridoclax to 25 µM leads to
comparable results in ellipsometry and in surface pressure
measurement (Figure 5). However, the decrease of surface
pressure and of the ellipsometric angle appear less pronounced,
suggesting a more efficient insertion of the Pyridoclax when used
at higher concentration. Moreover, as shown by AFM, the
organization of the phospholipid film in presence of 25 µM
Pyridoclax becomes different since the presence of spots with a
diameter around to 400 nm and a mean height of 40±3 nm are
homogeneously observed throughout the sample (Figure 6.B.).
The height could result of local aggregation of the amphipathic
molecule forming 3D structures.
The ability of Pyridoclax to induce the formation of phospholipids
aggregates contributes to a reorganization of the membrane, as
objectified by the CF leakage effect on LUVs formulated without
cholesterol. Such effect may contribute to the action mechanism
of this anticancer lead.
Figure 5. Ellipsometry (empty symbols) and surface pressure (full symbols)
kinetic measurements performed on MR29072 in presence of a DOPC:DOPG
film spread at the air-buffer (HEPES 50 mM + NaCl 107 mM) interface at initial
surface pressure of 30 mN/m at two concentrations 10 µM (ring), and 25 µM
(diamond).
10
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10.1002/cmdc.201900542
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0.30 mmol) were added 3-pyridylboronic acid (0.08 g, 0.66 mmol, 2.2
equiv), tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.03 mmol, 10%
mol) and aqueous K3PO4 (0.32 g, 0.15 mmol, 5.0 equiv) in 1,4-dioxane (60
mL). The mixture was stirred at reflux for 2 h, followed by TLC. The mixture
was cooled to room temperature, quenched with water and extracted with
ethyl acetate (3 x 100 mL). The organic layers were collected, washed with
brine, dried over anhydrous magnesium sulfate, filtered and concentrated
under reduced pressure to give the crude compound that was purified by
flash chromatography on silica gel (CH2Cl2/Methanol 99/1) to afford 0.09
g of pure compound MR33716 as a light yellow solid (57% yield). Mp: 162–
164 °C. 1H NMR (CDCl3) δ 9.27 (dd, J = 2.3, 0.9 Hz, 1H), 9.02 (dd, J = 2.3,
0.9 Hz, 1H), 8.97 (dd, J = 2.3, 0.9 Hz, 1H), 8.88 (dd, J = 2.1, 0.6 Hz, 1H),
8.87 (d, J = 2.2 Hz, 1H), 8.71 (dd, J = 4.8, 1.6 Hz, 1H), 8.68 (dd, J = 4.8,
1.7 Hz, 1H), 8.40 (ddd, J = 7.9, 2.4, 1.7 Hz, 1H), 8.24 (d, J = 2.2 Hz, 1H),
8.12 (dd, J = 8.1, 2.3 Hz, 1H), 8.05 (dd, J = 2.2, 0.8 Hz, 1H), 8.01 (ddd, J
= 7.9, 2.4, 1.6 Hz, 1H), 7.90 (dd, J = 8.2, 0.9 Hz, 1H), 7.49–7.46 (m, 3H),
7.44 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 7.36 (t, J = 7.4 Hz, 2H), 7.30 (m, 1H),
7.29 (s, 1H), 7.25 (s, 1H), 2.56 (s, 3H). 13C NMR (CDCl3) δ 155.0, 154.4,
153.5, 150.4, 150.2, 149.7, 148.6, 148.4, 148.3, 147.1, 140.1, 137.9, 136.5,
135.1, 134.6, 134.6, 134.3, 133.3, 133.2, 133.1, 132.8, 132.2, 131.4, 129.0,
128.7, 127.0, 124.8, 124.0, 123.8, 120.2, 20.3. MS (ESI+): m\z 504.49 (M
+ H+), 505.53 (M + 2H+). HRMS (ES): m/z calcd for C34H25N5 [M+H]+
504.2188, found 504.2191.
Conclusions
The experimental data generated in the present study on 19
original oligopyridines have permitted to assess relative
differences in terms of physico-chemical properties, which could
be determinant for their ADME-Tox properties. By this way, the
druggability pattern of these oligopyridines of therapeutic interest
as potential protein-protein interactions disruptors, and in
particular, those of the lead of this family, is more informed:
druggability space seems to be limited to 4 aromatic rings,
associated to styryl group on
a pyridine. Possible direct
interactions between the compounds and biological membranes
should be considered when their action mechanism will be
explored. Further studies are now to plan for detailing in deep the
high potential of this library of oligopyridines, being the presented
result encouraging and considered as a settled part of the drug
discovery and characterization work.
Experimental Section
Chemical Synthesis
Commercial reagents were used without additional purification. Only new
compounds are described. H NMR (400 MHz) and 13C NMR (100 MHz)
5-Bromo-2-[6-(6-iodo-3-pyridyl)-5-methyl-3-pyridyl]-3-[(E)-styryl]pyridine
terpyridine 2
1
To a microwave vial containing 2-(6-bromo-3-pyridyl)-5-[5-bromo-3-[(E)-
styryl]-2-pyridyl]-3-methylpyridine 1 (0.28 g, 0.56 mmol) were added NaI
(0.84 g, 5.60 mmol, 10.0 equiv) and CH3COCl (0.09 g, 1.12 mmol, 2.0
equiv) in acetonitrile (15 mL). The halogen exchange was carried out under
microwave irradiation at 100 °C for 1 h (400 W). The reaction was carefully
quenched by addition of saturated aqueous NaHCO3 solution until pH 8.
The solution was extracted with ethyl acetate (3 x 100 mL) and washed
with saturated aqueous NaHSO3 solution. The organic layers were
collected, washed with brine, dried over anhydrous magnesium sulfate,
filtered and concentrated under reduced pressure to give 0.29 g of the
crude compound 2 (94% yield, calculated by 1H NMR) as yellow solid that
can be used in the next reaction without further purification. 1H NMR
(CDCl3) δ 8.77 (d, J = 1.9 Hz, 1H), 8.68 (d, J = 2.1 Hz, 1H), 8.64 (d, J =
2.3 Hz, 1H), 8.19 (d, J = 2.1 Hz, 1H), 7.93 (d, J = 1.6 Hz, 1H), 7.85 (d, J =
8.1 Hz, 1H), 7.62 (dd, J = 8.1, 2.5 Hz, 1H), 7.43 (m, 2H), 7.37 –7.28 (m,
4H), 7.10 (q, J = 16.2 Hz, 2H), 2.45 (s, 3H). 13C NMR (CDCl3) δ 153.90,
153.47, 150.81, 148.56, 148.19, 141.10, 140.03, 139.40, 136.65, 136.23,
135.33, 134.50, 133.94, 133.12, 131.09, 129.01, 128.90, 127.91, 127.05,
119.98, 117.50, 19.90. MS (ESI+): m\z 554.36 (M + H+), 556.33 (M+ + 2),
557.32 (M+ + 3).
spectra were recorded on a Bruker 400 spectrometer. Chemical shifts are
expressed in parts per million downfield from tetramethylsilane as an
internal standard and coupling constants in hertz. Chemical shifts are
reported in parts per million (ppm) relative to the solvent resonance.
Melting points were recorded using a Kofler bench. Chromatography was
carried out on a column using flash silica gel 60 Merck (0.063 - 0.200 mm)
as the stationary phase. The eluting solvent indicated for each purification
was determined by thin layer chromatography (TLC) performed on 0.2 mm
precoated plates of silica gel 60F-264 (Merck) and spots were visualized
using an ultraviolet-light lamp. Purities of samples subjected to biological
testing were assessed using these two methods and shown to be ≥95%.
High resolution mass spectra were performed at 70 eV by electronic
impact (HRMS/EI) or by positive or negative electrospray (HRMS/ESI) on
a Xevo® G2-XS QTof (Waters).
General procedure A for Suzuki-Miyaura coupling reactions: 2-(6-Bromo-
3-pyridyl)-5-[5-bromo-3-[(E)-styryl]-2-pyridyl]-3-methyl-pyridine 1
To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-(6-
iodo-5-methyl-3-pyridyl)-3-[(E)-styryl]pyridine, obtained as reported in the
literature,[4] (0.37 g, 0.78 mmol) were added 6-bromopyridin-3-ylboronic
acid
(0.17
g,
0.85
mmol,
1.1
equiv),
tetrakis-
2-[6-(6-Bromo-3-pyridyl)-3-pyridyl]-5-[5-bromo-3-[(E)-styryl]-2-pyridyl]-3-
methyl-pyridine 3
(triphenylphosphine)palladium(0) (0.05 g, 0.04 mmol, 5% mol) and
aqueous K3PO4 (0.41 g, 1.9 mmol, 2.5 equiv) in 1,4-dioxane (80 mL). The
mixture was stirred at reflux for 3 h, followed by TLC. The mixture was
cooled to room temperature, quenched with water and extracted with ethyl
acetate (3 x 100 mL). The organic layers were collected, washed with brine,
dried over anhydrous magnesium sulfate, filtered and concentrated under
reduced pressure to give the crude compound that was purified by column
chromatography (c-hexane/ethyl acetate 4/1) to afford 0.28 g of pure
compound 1 as a yellow solid (72% yield). Mp: 174–178 °C. 1H NMR
(CDCl3) δ 8.77 (d, J = 1.7 Hz, 1H), 8.69 (d, J = 2.0 Hz, 1H), 8.65 (d, J =
2.3 Hz, 1H), 8.21 (d, J = 2.0 Hz, 1H), 7.94 (d, 1H), 7.85 (dd, J = 8.2, 2.4
Hz, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 7.2 Hz, 2H), 7.40–7.30 (m,
3H), 7.17–7.04 (m, 2H), 2.48 (s, 3H). 13C NMR (CDCl3) δ 154.2, 152.1,
150.4, 149.6, 148.2, 142.0, 140.0, 139.3, 136.6, 136.2, 135.1, 134.14,
133.9, 133.6, 131.3, 129.0, 128.9, 127.9, 127.1, 123.7, 120.5, 20.1. MS
(ESI+): m\z 508.39 (M + H+); 510.41 (M+ + 2).
To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-[6-(6-
iodo-3-pyridyl)-5-methyl-3-pyridyl]-3-[(E)-styryl]pyridine 2 (0.29 g, 0.53
mmol) were added 6-bromopyridin-3-ylboronic acid (0.12 g, 0.58 mmol,
1.1 equiv), tetrakis(triphenylphosphine)palladium(0) (0.03 g, 0.03, mmol
5% mol) and aqueous K3PO4 (0.28 g, 1.33 mmol, 2.5 equiv) in 1,4-dioxane
(50 mL) and the procedure A was performed. The mixture was stirred at
reflux for 3 h following by TLC the consumption of starting material. The
residue was purified by chromatography on silica gel (c-hexane/ethyl
acetate = 4/1) to afford 0.13 g of pure compound 3 as a yellow solid (42%
yield). mp: 206–208 °C. 1H NMR (CDCl3) δ 9.02 (d, J = 2.0 Hz, 1H), 8.99
(d, 1H), 8.81 (d, 1H), 8.70 (d, J = 2.0 Hz, 1H), 8.28 (dd, J = 8.3, 2.5 Hz,
1H), 8.22 (d, J = 1.8 Hz, 1H), 8.11 (dd, J = 8.2, 2.1 Hz, 1H), 7.97 (d, J =
1.8 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.45 (d, J =
7.2 Hz, 2H), 7.40–7.24 (m, 3H), 7.22–7.07 (m, 2H), 2.57 (s, 3H). 13C NMR
(CDCl3) δ 154.8, 153.0, 152.2, 150.4, 149.6, 148.6, 148.1, 142.8, 139.9,
137.9, 136.9, 136.6, 136.2, 135.3, 133.9, 133.8, 133.5, 131.3, 128.9, 128.8,
128.2, 127.0, 123.7, 120.4, 119.9, 20.2. MS (ESI+): m\z 585.36 (M + H+),
587.38 (M+ + 2).
General procedure for Suzuki-Miyaura coupling (B): 2-[5-Methyl-6-[6-(3-
pyridyl)-3-pyridyl]-3-pyridyl]-5-(3-pyridyl)-3-[(E)-styryl]pyridine (MR33716)
To a reaction vessel in a nitrogen atmosphere containing 2-(6-bromo-3-
pyridyl)-5-[5-bromo-3-[(E)-styryl]-2-pyridyl]-3-methyl-pyridine 1 (0.15 g,
11
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10.1002/cmdc.201900542
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2-[5-Methyl-6-[6-[6-(3-pyridyl)-3-pyridyl]-3-pyridyl]-3-pyridyl]-5-(3-pyridyl)-
3-[(E)-styryl] pyridine (MR33715)
To a reaction vessel in a nitrogen atmosphere containing 2-[6-(6-bromo-3-
(E)-2-Chloro-3-styrylpyridine 4
To a reaction vessel in a nitrogen atmosphere containing 3-iodo-2-
chloropyridine (0.92 g, 3.80 mmol) were added (E)-styrylboronic acid (1.1
equiv, 0.62 g, 4.20 mmol), tetrakis(triphenylphosphine)palladium(0) (0.22
g, 0.19 mmol, 5% mol) and aqueous K3PO4 (2.5 equiv, 2.03 g, 9.56 mmol)
in dioxane (200 mL) and the procedure A was performed. The mixture was
stirred at reflux for 3h following by TLC the consumption of starting material.
The residue was purified by chromatography on silica gel
(cyclohexane/ethyl acetate = 99/1) to afford 0.366 g of pure compound as
a white solid (52% yield). Mp: 94–96 °C. 1H NMR (CDCl3) δ 8.29 (dd, J =
4.7, 1.8 Hz, 1H), 7.97 (dd, J = 7.7, 1.8 Hz, 1H), 7.55 (d, J = 7.3 Hz, 2H),
7.45–7.30 (m, 3H), 7.25 (dd, J = 7.7, 4.7 Hz, 1H), 7.09 (d, J = 16.3 Hz, 1H);
13C NMR (CDCl3) δ 150.3, 148.2, 136.5, 134.7, 133.4, 132.3, 129.0, 128.8,
127.1, 123.2, 122.9. MS (ESI+): m\z 216.38 (M + H+), 218.35 (M+ + 2).
pyridyl)-3-pyridyl]-5-[5-bromo-3-[(E)-styryl]-2-pyridyl]-3-methyl-pyridine
3
(0.13 g, 0.22 mmol) were added 3-pyridylboronic acid (0.06 g, 0.48 mmol,
2.2 equiv), tetrakis(triphenylphosphine)palladium(0) (0.03 g, 0.02 mmol,
10% mol) and aqueous K3PO4 (0.23 g, 0.11 mmol, 5.0 equiv) in 1,4-
dioxane (50 mL) and the procedure B was performed. The mixture was
stirred at reflux for 3 h following by TLC the consumption of starting
material. The residue was purified by flash chromatography on silica gel
(CH2Cl2 /Methanol 99/1) to afford 0.07 g of pure compound MR33715 as
1
a white solid (51% yield). Mp: 240 °C. H NMR (CDCl3) δ 9.39 (d, J = 2.2
Hz, 1H), 9.30 (s, 1H), 9.05 (d, J = 2.2 Hz, 1H), 8.99 (d, J = 2.4 Hz, 1H),
8.90 (d, J = 2.2 Hz, 1H), 8.89 (d, J = 2.2 Hz, 1H), 8.73 (dd, J = 4.8, 1.6 Hz,
1H), 8.76–8.67 (m, 1H), 8.55 (dd, J = 8.2, 2.3 Hz, 1H), 8.43 (dt, J = 8.0,
1.9 Hz, 1H), 8.26 (d, J = 2.2 Hz, 1H), 8.16 (dd, J = 8.1, 2.3 Hz, 1H), 8.07
(d, J = 2.1 Hz, 1H), 8.03 (dt, J = 7.9, 1.9 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H),
7.92 (d, J = 8.3 Hz, 1H), 7.51–7.48 (m, 3H), 7.47–7.44 (m, 1H), 7.40–7.35
(m, 2H), 7.33–7.29 (m, 1H), 7.28 (s, 1H), 7.27 (s, 1H), 2.59 (s, 3H). 13C
NMR (CDCl3) δ 155.1, 154.9, 153.8, 153.5, 150.4, 150.2, 149.7, 148.6,
148.4, 148.3, 148.3, 147.0, 140.0, 137.8, 136.4, 135.5, 135.1, 134.5, 134.4,
134.3, 133.6, 133.2, 133.1, 133.0, 132.7, 132.1, 131.3, 128.9, 128.6, 126.9,
124.7, 123.9, 123.7, 120.5, 120.0, 20.2. MS (ESI+): m\z 581.60 (M + H+),
582.63 (M + 2H+). HRMS (ES): m/z calcd for C39H28N6 [M+H]+ 581.2454,
found 581.2462.
(E)-5'-Methyl-3-styryl-2,3'-bipyridine (MR33717)
To a reaction vessel in a nitrogen atmosphere containing (E)-2-chloro-3-
styrylpyridine 4 (0.37 g, 1.69 mmol) were added 5-methylpyridin-3-
ylboronic
acid
(1.5
equiv,
0.35
g,
2.53
mmol),
tetrakis(triphenylphosphine)palladium(0) (0.098 g, 0.08 mmol, 5% mol)
and aqueous K3PO4 (0.897 g, 4.22 mmol, 2.5 equiv) in 1,4-dioxane (40
mL) and the procedure A was performed. The mixture was stirred at reflux
for 3 h following by TLC the consumption of starting material. The residue
was purified by chromatography on silica gel (c-hexane/ethyl acetate 7/3)
to afford 0.30 g of pure compound MR33717 as a beige oil (65% yield). 1H
NMR (CDCl3) δ 8.71 (d, J = 1.8 Hz, 1H), 8.66 (dd, J = 4.7, 1.6 Hz, 1H),
8.56 (m, 1H), 8.07 (dd, J = 8.0, 1.5 Hz, 1H), 7.86 (m, 1H), 7.45 (d, J = 1.5
Hz, 1H), 7.43 (s, 1H), 7.39–7.33 (m, 3H), 7.32–7.29 (m, 1H), 7.14 (s, 2H),
2.45 (s, 3H). 13C NMR (CDCl3) δ 154.5, 150.0, 148.7, 147.8, 137.8, 136.8,
135.2, 134.2, 133.1, 132.2, 131.8, 128.9, 128.4, 126.9, 125.2, 123.1, 18.6.
MS (ESI+): m\z 273.54 (M + H+), 274.53 (M + 2H+). HRMS (ES): m/z calcd
for C19H16N2 [M+H]+ 273.1392, found 273.1403.
2,5-Bis(3-pyridyl)-3-[(E)-styryl]pyridine (MR29063)
To a reaction vessel in a nitrogen atmosphere containing (E)-5-bromo-2-
chloro-3-styrylpyridine (obtained as reported in Gloaguen et al.[4]) (0.40 g,
1.4 mmol) were added 3-pyridylboronic acid (0.42 g, 3.40 mmol, 2.5 equiv),
tetrakis(triphenylphosphine)palladium(0) (0.16 g, 0.14 mmol, 10% mol)
and aqueous Na2CO3 (0.72 g, 6.5 mmol, 5.0 equiv,) in 1,4-dioxane (150
mL) and the procedure B was performed. The mixture was stirred at reflux
for 20 h following by TLC the consumption of starting material. The residue
was purified by chromatography on silica gel (CH2Cl2 /Methanol 98/2) to
afford 0.28 g of pure compound MR29063 as a white solid (61% yield).
Mp: 119 °C. 1H NMR (CDCl3) δ 8.96 (d, J = 1.9 Hz, 1H), 8.95 (d, J = 1.9
Hz, 1H), 8.85 (d, J = 1.9 Hz, 1H), 8.70 (d, J = 4.0 Hz, 2H), 8.21 (d, J = 1.9
Hz, 1H), 8.04 (dt, J = 7.8, 1.9 Hz, 1H), 7.99 (dt, J = 7.8, 1.9 Hz, 1H), 7.48
(m, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.35 (t, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8
Hz, 1H), 7.21 (d, J = 16.6 Hz, 1H), 7.14 (d, J = 16.6 Hz, 1H). 13C NMR
(CDCl3) δ 153.8, 150.5, 149.6, 149.5, 148.1, 146.8, 137.1, 136.3, 134.9,
134.4, 133.0, 132.9, 132.8, 132.5, 131.9, 128.8, 128.5, 126.8, 124.6, 123.8,
123.2. MS (ESI+): m\z 336.58 (M + H+), 337.57 (M + 2H+). HRMS (ES):
m/z calcd for C23H17N3 [M+H]+ 336.4123, found 336.2298.
2-Bromo-5-(5-bromo-3-methyl-2-pyridyl)-3-methyl-pyridine 5
To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-iodo-
3-methyl-pyridine (0.700 g, 2.3 mmol) were added 6-bromo-5-methyl-3-
pyridylboronic
acid
(0.56
g,
2.80
mmol,
1.3
equiv)
tetrakis(triphenylphosphine)palladium(0) (0.13 g, 0.12 mmol, 5% mol) and
aqueous Na2CO3 (0.62 g, 5.90 mmol, 2.5 equiv) in 1,4-dioxane (10 mL)
and the procedure B was performed. The residue was purified by
chromatography on silica gel (c-hexane/ethyl acetate 95/5) to afford 0.62
g of pure compound 5 as a yellow solid (77% yield). Mp: 130 °C. 1H NMR
(CDCl3) δ 8.60 (d, J = 1.9 Hz, 1H), 8.35 (d, J = 1.9 Hz, 1H), 7.78 (d, J =
1.9 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 2.46 (s, 3H), 2.38 (s, 3H). 13C NMR
(CDCl3) δ 152.8, 148.5, 147.0, 144.5, 141.1, 139.2, 135.0, 134.5, 133.0,
119.9, 22.0, 19.8. MS (ESI+): m\z 342.31 (M + H+), 344.78 (M+ + 2).
3'-Methyl-3,2':5',3''-terpyridine (MR33714)
To a reaction vessel in a nitrogen atmosphere containing 2,5-dibromo-3-
methylpyridine (3.0 g, 12.7 mmol) were added 3-pyridylboronic acid (3.11
g, 25.30 mmol, 2.0 equiv), tetrakis(triphenylphosphine)palladium(0) (1.46
g, 1.27 mmol, 10% mol) and aqueous K2CO3 (8.73 g, 63.30 mmol, 5.0
equiv) in 1,4-dioxane (250 mL) and the procedure A was performed. The
mixture was stirred at reflux for 24 h following by TLC the consumption of
starting material. The mixture was cooled at room temperature and
quenched with water and extracted with ethyl acetate (3 x 200 mL). The
crude compound was filtered on Gooch and washed with 100 mL of c-
hexane/ethyl acetate 6/4 solution, affording 2.75 g of pure compound
MR33714 as a light brown solid (65% yield). Mp: 152–156 °C. 1H NMR
(400 MHz, CDCl3) δ 8.90 (d, J = 1.8 Hz, 1H), 8.86 (d, J = 1.8 Hz, 1H), 8.79
(d, J = 2.2 Hz, 1H), 8.68 (dd, J = 4.7, 1.0 Hz, 2H), 7.97–7.92 (m, 2H), 7.83
(d, J = 2.2 Hz, 1H), 7.47–7.42 (m, 2H), 2.17 (s, 3H). 13C NMR (CDCl3) δ
155.2, 149.9, 149.5, 149.3, 148.2, 145.7, 137.2, 136.5, 135.7, 134.4, 133.1,
132.6, 131.5, 123.8, 123.3, 20.1. MS (ESI+): m\z 248.45 (M + H+), 249.53
(M + 2H+). HRMS (ES): m/z calcd for C16H13N3 [M+H]+ 248.1188, found
248.1190.
5-Bromo-2-(6-iodo-5-methyl-3-pyridyl)-3-methyl-pyridine 6
To a reaction vessel containing 2-bromo-5-(5-bromo-3-methyl-2-pyridyl)-
3-methyl-pyridine 5 (1.8 g, 5.3 mmol) were added NaI (1.97 g, 13.10 mmol,
2.5 equiv) and CH3COCl (0.62 g, 7.90 mmol, 1.5 equiv) in acetonitrile (100
mL). The mixture was stirred at reflux for 4 h, cooled at room temperature
quenched with water and extracted with ethyl acetate (3 x 100 mL). The
organic layers were collected, washed with brine, dried over anhydrous
magnesium sulfate, filtered and concentrated under reduced pressure to
give the crude compound which was put in the reaction conditions
described upon and stirred at reflux for 4 h. The reaction was carefully
quenched by addition of saturated aqueous NaHCO3 solution until pH–8.
The solution was extracted with ethyl acetate (3 x 100 mL) and washed
with saturated aqueous NaHSO3 solution. The organic layers were
collected, washed with brine, dried over anhydrous magnesium sulfate,
filtered and concentrated under reduced pressure to give 0.29 g of the
crude compound which was purified by chromatography on silica gel (c-
hexane/ethyl acetate 9/1) to afford 1.91 g of pure compound 6 as a beige
solid (93% yield). Mp: 128 °C. 1H NMR (CDCl3) δ 8.58 (d, J = 1.9 Hz, 1H),
8.31 (d, J = 1.9 Hz, 1H), 7.77 (d, J = 1.9 Hz, 1H), 7.62 (d, J = 1.9 Hz, 1H),
2.46 (s, 3H), 2.38 (s, 3H). 13C NMR (CDCl3) δ 152.9, 148.5, 147.4, 141.1,
12
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10.1002/cmdc.201900542
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FULL PAPER
139.1, 137.1, 134.7, 133.0, 125.0, 119.9, 26.2, 19.8. MS (ESI+): m\z
under reduced pressure to give the crude product which was recrystallized
from diethyl ether give 1.17 g of pure compound 9 as white solid (68%).
Mp: 184 °C. 1H NMR (CDCl3) δ 8.79 (d, J = 2.0, 1H), 8.50 (dd, J = 5.0, 1.5,
1H), 8.09 (dt, J = 7.5, 1.5, 1H), 7.51 (dd, J = 8.0, 5.0, 1H), 7.49 (s, 1H),
7.46 (d, J = 8.0, 1H), 7.40 (d, J = 8.0, 1H), 2.42 (s, 3H). 13C NMR (CDCl3)
δ 148.7, 148.3, 142.3, 138.8, 138.7, 136.5, 135.4, 133.3, 128.8, 125.5,
124.6, 22.1. MS (ESI+): m\z 214.45 (M + H+), 215.48 (M+ + 2H+).
389.36 (M + H+), 392.31 (M+ + 2).
5-Bromo-2-[6-(6-bromo-5-methyl-3-pyridyl)-5-methyl-3-pyridyl]-3-methyl-
pyridine 7
To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-(6-
iodo-5-methyl-3-pyridyl)-3-methyl-pyridine 6 (1.7 g, 4.40 mmol) were
added 6-bromo-5-methyl-3-pyridylboronic acid (1.13 g, 5.20 mmol, 1.3
equiv), tetrakis(triphenylphosphine)palladium(0) (0.25 g, 0.22 mmol, 5%
mol) and aqueous Na2CO3 (1.16 g, 10.90 mmol, 2.5 equiv) in 1,4-dioxane
(150 mL) and the procedure A was performed. The residue was purified
by chromatography on silica gel (c-hexane/ethyl acetate 9/1) to afford 1.40
g of pure compound 7 as a yellow solid (75% yield). Mp: 141 °C. 1H NMR
(CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H), 8.42 (d, J =
5-Bromo-2-(pyridin-3-yl)benzaldehyde 10
To
dibromobenzaldehyde (2.0 g, 7.58 mmol) were added 3-pyridylboronic
acid (1.16 g, 9.47 mmol, 1.3 equiv), tetrakis-
a reaction vessel in a nitrogen atmosphere containing 2,5-
(triphenylphosphine)palladium(0) (0.44 g, 0.38 mmol, 5% mol) and
aqueous Na2CO3 (2.0 g, 18.94 mmol, 2.5 equiv) in 1,4-dioxane (100 mL)
following procedure A. After cooling down to room temperature, the
mixture was filtered on Celite and the pad was washed with CH2Cl2 (300
mL). The organic layers were collected, washed with brine, dried over
anhydrous magnesium sulfate, filtered and concentrated under reduced
pressure to give the crude product which was purified by chromatography
on silica gel (c-hexane/ethyl acetate 9/1) to afford 1.6 g of pure compound
10 as a white solid (80% yield). Mp: 140 °C. 1H NMR (CDCl3) δ 9.90 (s,
1H), 8.72 (dd, J = 4.9, 2.0, 1H), 8.65 (s, 1H), 8.19 (d, J = 2.0, 1H), 7.81 (dd,
J = 7.8, 2.0, 1H), 7.70 (dd, J = 7.8, 2.0, 1H), 7.44 (dd, J = 7.8, 4.9, 1H),
7.32 (d, J = 7.8, 1H). 13C NMR (CDCl3) δ 189.7, 149.9 (2C), 149.7, 140.4,
137.0, 136.7, 134.9, 132.5, 131.2, 123.2, 123.1. MS (ESI+): 262.39 (M +
H+), 264.40. (M+ + 2).
1.9 Hz, 1H), 7.79‒7.81 (m, 3H), 2.48 (s, 3H), 2.46 (s, 3H), 2.44 (s, 3H). 13
C
NMR (CDCl3) δ 153.9, 153.5, 148.5, 147.3, 147.2, 144.3, 141.0, 139.4,
139.3, 135.2, 134.9, 134.5, 133.1, 131.0, 119.8, 22.0, 20.0, 19.9. MS
(ESI+): m\z 434.52 (M + H+), 436.44 (M+ + 2).
3-Methyl-2-[5-methyl-6-[5-methyl-6-(3-pyridyl)-3-pyridyl]-3-pyridyl]-5-(3-
pyridyl)pyridine (MR29015)
To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-[6-(6-
bromo-5-methyl-3-pyridyl)-5-methyl-3-pyridyl]-3-methyl-pyridine 7 (0.39 g,
0.90 mmol) were added 3-pyridylboronic acid (0.29 g, 2.3 mmol, 2.5 equiv),
tetrakis(triphenylphosphine)palladium(0) (0.10 g, 0.09 mmol, 10% mol)
and aqueous Na2CO3 (5.0 equiv, 0.48 g, 4.60 mmol) in 1,4-dioxane (80
mL) and the procedure B was performed. The residue was purified by
chromatography on silica gel (CH2Cl2 /Methanol 98/2) to afford 0.29 g of
pure compound MR29015 as a white solid (74% yield). Mp: 193 °C. 1H
NMR (CDCl3) δ 8.92 (d, J = 1.9 Hz, 1H), 8.88 (d, J = 1.9 Hz, 1H), 8.82 (d,
J = 1.9 Hz, 3H), 8.69 (dd, J = 7.8, 1.9 Hz, 1H), 8.68 (d, J = 1.9 Hz, 1H),
7.96 (m, 2H), 7.93 (d, J = 1.9 Hz, 2H), 7.85 (d, J = 1.9 Hz, 1H), 7.45 (m,
2H), 2.57 (s,3H), 2.56 (s,3H), 2.49 (s,3H). 13C NMR (CDCl3) δ 154.9, 154.7,
154.6, 149.9, 149.4, 149.1, 148.2, 147.4, 147.3, 145.7, 139.3 (2C), 137.2,
136.5, 135.9, 135.0, 134.8, 134.3, 133.0, 132.6, 131.5, 131.1, 131.0, 123.8,
123.1, 20.1, 20.0, 19.9. MS (ESI+): m\z 430.71 (M + H+), 431.65 (M+ 2H+).
HRMS (ES): m/z calcd for C28H23N5 [M+H]+ 430.1953, found 430.1961.
2-(Pyridin-3-yl)-5-(2-methyl-4-pyridin-3-ylphenyl)benzaldehyde 11
To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-
(pyridin-3-yl)benzaldehyde 10 (0.500 g, 1.91 mmol) were added 2-methyl-
4-(pyridin-3-yl)phenylboronic acid 9 (0.51 g, 2.38 mmol, 1.3 equiv),
tetrakis(triphenylphosphine)palladium(0) (0.11 g, 0.10 mmol, 5% mol) and
aqueous K3PO4 (1.01 g, 4.77 mmol, 2.5 equiv) in dimethoxyethane (80
mL). The mixture was stirred at reflux 18 h following by TLC the
consumption of starting material. After cooling down to room temperature,
the mixture was filtered on Celite and the pad was washed with CH2Cl2
(100 mL). The organic layers were collected, washed with brine, dried over
anhydrous magnesium sulfate, filtered and concentrated under reduced
pressure to give the crude product which was purified by chromatography
on silica gel (c-hexane/ethyl acetate 1/1) to afford 0.62 g of pure compound
3-(4-Bromo-3-methylphenyl)pyridine 8
To a reaction vessel in a nitrogen atmosphere containing 2-bromo-5-
iodotoluene (1.0 g, 3.36 mmol) were added 3-pyridylboronic acid (1.3 equiv,
0.52 g, 4.21 mmol), tetrakis(triphenylphosphine)palladium(0) (0.19 g, 0.17
mmol, 5% mol) and aqueous Na2CO3 (0.89 g, 8.42 mmol, 2.5 equiv) in 1,4-
dioxane (150 mL) and procedure A was performed. After cooling down to
room temperature, the mixture was filtered on Celite and the pad was
washed with CH2Cl2 (150 mL). The organic layers were collected, washed
with brine, dried over anhydrous magnesium sulfate, filtered and
concentrated under reduced pressure to give the crude product which was
purified by chromatography on silica gel (c-hexane/ethyl acetate 9/1) to
afford 0.71 g of pure compound 8 as a yellow oil (85% yield). 1H NMR
(CDCl3) δ 8.80 (d, J = 2.9, 1H), 8.60 (d, J = 4.9, 1H), 7.83 (dt, J = 7.8, 2.0,
1H), 7.62 (d, J = 8.8, 1H), 7.43 (d, J = 2.0, 1H), 7.35 (dd, J = 7.8, 4.9, 1H),
7.25 (dd, J = 8.8, 2.0, 1H), 2.47 (s, 3H). 13C NMR (CDCl3) δ 148.7, 148.1,
138.7, 137.0, 135.7, 134.2, 133.0, 129.5, 126.0, 125.0, 123.6, 23.1. MS
(ESI+): m\z 248.37 (M + H+), 250.39 (M+ + 2), 251.40 (M+ + 3).
1
11 as a white solid (93% yield). Mp: 162 °C. H NMR (CDCl3) δ 10.06 (s,
1H), 8.75 (s, 1H), 8.73 (s, 1H), 8.63 (d, J = 4.6, 2H), 8.08 (d, J = 2.0, 1H),
7.93 (dt, J = 8.0, 1.7, 1H), 7.79 (dd, J = 8.0, 1.9, 1H), 7.71 (dd, J = 7.8, 2.0,
1H), 7.54‒7.48 (m, 3H), 7.51‒7.45 (m, 1H), 7.42‒7.38 (m, 2H), 2.42 (s,
3H). 13C NMR (CDCl3) δ 191.2, 150.1, 149.4, 148.6, 148.3, 141.9, 140.4,
139.7, 137.5, 137.2, 136.1, 136.0, 134.5, 134.3, 133.6, 133.4, 131.0, 130.5,
129.3, 129.0, 124.8, 123.6, 123.2, 20.6. MS (ESI+): m\z 351.50 (M + H+),
352.44 (M + 2H+).
2-(Pyridin-3-yl)-5-(2-methyl-4-pyridin-3-ylphenyl)-(E)styrylphenyl
(MR30821)
To a stirred solution of diethyl benzylphosphonate (0.07 g, 0.28 mmol, 1.0
equiv) in THF (20 mL) under nitrogen flow was added KOH (0.03 g, 0.57
mmol, 2.0 equiv). After 30 min of stirring, 2-(pyridin-3-yl)-5-(2-methyl-4-
pyridin-3-ylphenyl)benzaldehyde 11 (0.10 g, 0.28 mmol) was added. The
mixture was stirred at room temperature for 1 h and then refluxed 5 h until
the starting material was consumed (TLC). The mixture was cooled to
room temperature, quenched with water and extracted with ethyl acetate
(3 x 50 mL). The organic layers were collected, washed with brine, dried
over anhydrous magnesium sulfate, filtered and concentrated under
reduced pressure to give the crude compound that was purified by column
chromatography (c-hexane/ethyl acetate 1/1) affording 0.10 g of pure
compound MR30821 as a yellow solid (85%). Mp: 139 °C. 1H NMR (CDCl3)
δ 8.94 (s, 1H), 8.77 (s, 1H), 8.66 (s, 1H), 8.61 (s, 1H), 7.93 (d, J = 8.0, 1H),
7.79 (s, 1H), 7.55‒7.23 (m, 13H), 7.12 (s, 2H), 2.47 (s, 3H). 13C NMR
(CDCl3) δ 150.2, 148.3 (2C), 148.1, 141.2, 141.0, 137.0, 136.9, 136.1 (2C),
135.8, 135.5, 134.1 (2C), 130.7, 130.4, 130.1, 129.1, 128.6 (2C), 128.4,
127.7, 126.8, 126.5 (2C), 126.4, 124.5, 123.5, 122.9, 20.6. MS (ESI+): m\z
2-Methyl-4-(pyridin-3-yl)phenyl boronic acid 9
To a slurry solution of n-BuLi (4.03 mL, 10.07 mmol, 1.3 equiv) in
anhydrous THF (20 mL), cooled to -78°C, was added a solution 3-(4-
bromo-3-methylphenyl)pyridine 8 (2.0 g, 8.06 mmol) in THF (50 mL). The
mixture was allowed to react at this temperature for over 1 h. A solution of
triisopropylborate (2.32 mL, 10.07 mmol, 1.3 equiv) was then added and
left to react for 45 min. The mixture was allowed to warm to room
temperature and was quenched by slow addition of 1 M NaOH solution
(100 mL). The resulting aqueous layer was collected and acidified to pH–
7 by dropwise addition of 3 N HCl solution. The solution was extracted with
ethyl acetate (3 x 100 mL). The organic layers were collected, washed with
brine, dried over anhydrous magnesium sulfate, filtered and concentrated
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900542
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425.56 (M + H+), 426.63 (M + 2H+). HRMS (ES): m/z calcd for C31H24N2
[M+H]+ 425.2018, found 425.2018.
mixture of buffer with acetonitrinile/DMSO (99:1; vol/vol). Calibration
curves were linear with R2> 0.99.
5-Bromo-2-(3-pyridyl)pyridine 12
Permeability evaluation
To
a
reaction vessel in
a
nitrogen atmosphere containing 2,5-
The Pampa-GIT experiments were conducted using the Pampa Explorer
Kit (Pion Inc) according to manufacturer's protocol. Briefly, each stock
compound solution (20 mM in DMSO) was diluted in Prisma HT buffer pH
7.4 (pION) to 100 µM. 200 µL of these solutions (n=4) were added to the
donor plate (P/N 110243). 5 µL of the GIT-0 Lipid (P/N 110669) was used
to coat the membrane filter of the acceptor plate (P/N 110243). 200 µL of
the Acceptor Sink Buffer (P/N 110139) was added to each well of the
acceptor plate. The sandwich was incubated at room temperature for 4 h,
without stirring. After incubation, the UV-visible spectra were measured
with the microplate reader (Tecan infinite M200) and for each compound,
the -logPe and Pe were calculated by the PAMPA Explorer software v. 3.7
(pION). As recommended by the manufacturer, ketoprofen and antipyrin
were used as low (Pe = 8 nm/s at pH 7.4) and moderately (Pe = 16 nm/s
at pH 7.4) permeable references, respectively.
dibromopyridine (5.85 g, 24.6 mmol) were added 3-pyridylboronic acid
(3.79 g, 30.75 mmol, 1.3 equiv), tetrakis(triphenylphosphine)palladium(0)
(1.42 g, 1.23 mmol, 5% mol) and aqueous Na2CO3 (6.54 g, 61.50 mmol,
2.5 equiv) in 1,4-dioxane (300 mL) and procedure A was performed. The
crude product was purified by chomatography on silica gel (c-hexane/ethyl
acetate 4/1) to afford 3.70 g of pure compound 12 as a white solid (64%
yield). Mp: 78 °C. 1H NMR (CDCl3) δ 9.16 (dd, J = 2.3, 0.8 Hz, 1H), 8.76
(dd, J = 2.4, 0.6 Hz, 1H), 8.66 (dd, J = 4.8, 1.7 Hz, 1H), 8.29 (ddd, J = 8.0,
2.3, 1.7 Hz, 1H), 7.91 (dd, J = 8.4, 2.4 Hz, 1H), 7.65 (dd, J = 8.4, 0.7 Hz,
1H), 7.40 (ddd, J = 8.0, 4.8, 0.8 Hz, 1H). 13C NMR (CDCl3) δ 153.30, 151.24,
150.35, 148.11, 139.64, 134.23, 133.84, 123.72, 121.66, 120.28, 77.41,
77.30, 77.10, 76.78. MS (ESI+): m\z 235.42 (M + H+), 237.37 (M+ + 2).
2-(3-Pyridyl)-5-[6-(3-pyridyl)-3-pyridyl]pyridine (MR33718)
To a reaction vessel in a nitrogen atmosphere containing 5-bromo-2-(3-
pyridyl)pyridine 12 (0.25 g, 1.06 mmol) were added bispinacolato diboron
(0.14 g, 0.55 mmol, 0.52 equiv), PdCl2 (dppf) (0.09 g, 0.11 mmol, 10% mol)
and aqueous KOAc (0.26 g, 2.65 mmol, 2.5 equiv) in 1,4-dioxane (50 mL).
The mixture was refluxed 3 h, until the starting material was consumed
(TLC), cooled to room temperature, quenched with water and extracted
with CH2Cl2 (3 x 50 mL). The organic layers were collected, washed with
brine, dried over anhydrous magnesium sulfate, filtered and concentrated
under reduced pressure to give the crude compound that was purified by
column chromatography on silica gel (CH2Cl2/Methanol 98/2) to afford 0.09
g of pure compound MR33718 as a brown solid (56% yield). Mp: 240 °C.
1H NMR (CDCl3) δ 9.28 (d, J = 1.7 Hz, 2H), 9.03 (d, J = 1.8 Hz, 2H), 8.70
(dd, J = 4.7, 1.4 Hz, 2H), 8.41 (dt, J = 8.0, 1.9 Hz, 2H), 8.07 (dd, J = 8.2,
Experimental chrom logP determination
Chrom logP estimation was carried out using an isocratic liquid
chromatography method for basic compounds derived from method
described by Henchoz.[25] All experiments were performed on a UHPLC
Agilent 1290 Infinity system (Agilent Technologies, Santa Clara, California,
USA) equipped with a PDA detector 1260 operating at 220, 240, 254, 290
and 350 nm for all compounds. The chromatographic system was
controlled by Open LAB CDS LC Chemstation® software (revision C01.05).
Retention time measurement was performed at 27°C, with a flow rate 0.6
mL.min-1, and by using an Acquity BEH Shield RP18 column (1.7 µm, 2.1
x 50 mm) from Waters (Milford, MA, USA). Mobile phase was composed
by various percentage of methanol as organic modifier (35-85 %) and an
aqueous basic buffer (triethylammonium acetate solution at pH 11.5)
adjusted by sodium hydroxide addition) in order to keep calibration and
newly synthesized compounds in their neutral form.
Briefly, chrom logP was estimated by plotting the known logP[22] values of
9 standard compounds with their log k49%, which is the retention factor at
49% methanol in the mobile phase. Calculation of log k49% values was
performed from retention time (tR) measurement at three different mobile
phase compositions. Standard compounds were cimetidine, labetalol,
pindolol, papaverine, quinine, tetracaine, promethazine, chlorpromazine,
amiodarone which present a large range of log P values from 0.48 to 7.8.
Each compound was injected (1µL) once with each mobile phase
composition and tR determined from the apex of the peak. Log k49% values
were obtained by extrapolation to 49 % organic modifier using linear
relationships between the three log k values and methanol percentage
(r²>0.99 for all compounds).
2.4 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.46 (dd, J = 7.9, 4.8 Hz, 2H). 13
C
NMR (CDCl3) δ 154.51, 150.28, 148.27, 135.21, 134.30, 134.16, 132.07,
123.72, 120.64. MS (ESI+): m\z 311.54 (M + H+), 312.48 (M + 2H+). HRMS
(ES): m/z calcd for C20H14N4 [M+H]+ 311.1297, found 311.1301.
Physico-chemical studies
Calculated physico-chemical properties
Values of molecular weight, pKa, calc logP, the number of hydrogen-bond
acceptors, of rotatable bonds, of aromatic rings in scaffold, of lateral alkyl
substituents, and the polar surface area were calculated for the studied
molecules using standard tools of the ChemAxon Package (Marvin
16.1.4.0, 2016, ChemAxon (http://www.chemaxon.com/). Calc logP values
were also predicted using Discovery Studio,[55] Schrödinger,[56] and
Molinspiration (http://www.molinspiration.com/cgi-bin/properties/).
Then calibration curve was built with experimentally determined logP
values of standards compounds obtained from literature[22] vs their log k49%
Linear relationship between logP and calculated log k49% was expressed
by the following equation:
.
Thermodynamic solubility determination
Thermodynamic solubility at pH 7.4 of compounds was determined
according to the classical shake-flask method (Organisation for Economic
log k49% = 0.5226 x log P - 0.7058, with r2 = 0.978 and F = 444 [2]
Cooperation and Development guideline n° 105) and adapted from a
[57]
miniaturized procedure described elsewhere
. Phosphate Buffer
As described by Henchoz[25], the use of the Acquity BEH shield column
with a log k49% provides the best correlation results with experimental logP
values obtained with basic compounds.
solutions (pH 7.4, 10 μM, ionic strength 150 mM) were prepared from
Na2HPO4, KH2PO4 and KCl (Sigma Aldrich, Saint Quentin Fallavier,
France); 10 μL of 50 mM stock solution was added to 1.5 mL microtubes
(3 replicates) containing 990 mL buffer (n = 4). Tubes were briefly
sonicated and shacked by inversion during 24 h at room temperature.
Then, microtubes were centrifuged at 12,225 x g for 5 min; 100 μL
supernatant was mixed with 100 μL acetonitrile in a Greiner UV microplate.
Standard solutions were prepared extemporaneously diluting 50 mM
DMSO stock solutions at 0, 10 and 25 mM; 5 μL each working solution was
diluted with 995 μL buffer and 100 μL was then mixed in microplate with
100 μL acetonitrile to keep unchanged the final proportions of each solvent
in standard solutions and samples. Determination of solubility at pH 7.4
was made with a Synergy 2 (Biotek, Colmar, France) microplate reader in
spectrophotometric mode (230 to 450 nm) from the specific λmax of each
compound. The calibration curve obtained from the three standard
solutions of tested compounds at 0, 50, and 100 μM in a 50:50 (vol/vol)
Large Unilamellar Vesicles (LUVs) preparation and characterization
Two formulations of LUVs, with or without cholesterol (chol), have been
prepared according to the thin film hydration method and resized by
extrusion.[58] Briefly, phosphatidylcholine (PC from soybean, Lipoid S 100
was a gift of Lipoid GmbH, Ludwigshafen, Germany) and 1,2-dioleoyl-sn-
glycero-3-phospho-rac-1-glycerol sodium salt (DOPG-Na, Lipoid PG 18:1
kindly provided by Lipoid GmbH, Ludwigshafen, Germany) have been
mixed in a weight/weight (w/w) ratio of 4:1 (PC:DOPG) for the first
formulation of LUVs, with the addition of chol (Sigma-Aldrich, Saint-
Quentin Fallavier, France), in a w/w ratio of 4:1:1 (PC:DOPG:chol), for the
second one. Then, lipids mix has been dissolved in an adequate amount
of chloroform and after evaporation of organic solvent under a stream of
14
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10.1002/cmdc.201900542
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FULL PAPER
nitrogen, the dried film was kept overnight in a vacuum desiccator to
remove any residual trace of solvent. The resultant lipid film has been
hydrated with a buffer A (50 mM HEPES, 10 mM NaCl, 1 mM EDTA, pH
7.4) and the mixture mixed by vortexing and then stirred for 1 hour at room
temperature.
where I0 and I are the corrected fluorescence intensity of the probes (DPH
or TMA-DPH) in the absence and in presence of the drug, respectively.
Rifampicin (10 and 25 µM), known to be able to penetrate into membranes,
was used as reference.[52]
The fluorescence probes were directly dissolved in buffer B (50 mM
HEPES, 107 mM NaCl, 1 mM EDTA, pH 7.4) for carboxyfluorescein (CF,
11.6 mg/mL) or during the hydratation phase for 1,6-diphenyl-1,3,5-
hexatriene (DPH, 11.6 mg/mL) and 1-(4-trimethylammonium)-6-phenyl-
1,3,5-hexatriene (TMA-DPH, 11.6 mg/mL) in DMSO.
Lipid mixtures have been then extruded 13 times through polycarbonate
filters with a pore diameter of 100 nm and the obtained LUVs have been
separated from possible unincorporated material by passage through a
Sepharose CL-4B loaded column, using buffer B (50 mM HEPES, 107 mM
NaCl, pH 7.4) as eluent. The LUVs size and charge have been assessed
by Dynamic Light Scattering (DLS, NanoZS, Malvern Instruments,
Worcestershire, UK) after 1/100 dilution in buffer B. Granulometric
properties and -potential values of various formulations are presented in
Table S1 (see supporting information).
Ellipsometry and Surface Pressure Measurements
Experiments were performed with a computer-controlled and user-
programmable Langmuir Teflon®-coated trough (type 601BAM) equipped
with two movable barriers and of total surface 90 cm² (Nima Technology
Ltd., England). Before starting the experiments, the trough was cleaned
successively with ultrapure water (Nanopure-UV), ethanol, and finally
ultrapure water. The trough was filled with HEPES-buffer (50 mM HEPES,
107 mM NaCl, pH 7.4).
For lipid free interface or air/lipid interface experiments, the MR29072
solution was injected in the sub-phase using a Hamilton syringe to obtain
the final concentration of 10 µM or 25 µM.
To test the lipid/oligopyridine interactions, the lipid monolayers were
performed on the air/buffer interface by spreading the lipid solution with a
high precision Hamilton microsyringe to obtain an initial surface pressure
around 30 mN/m. This surface pressure value was selected because it is
known as similar to the in vivo membrane pressure.[59] After 10 min,
necessary to allow solvent evaporation, the oligopyridine solution was
injected in the subphase. The lipid solution concentration was 1 mg/mL
The entrapment of CF has been verified by dequenching of fluorescence
after the addition of 10 % (v/v) Triton X-100 measured with a Synergy 2
microplate reader (Biotek, Colmar, France) equipped with the appropriate
filters (λex = 485/20 nm and λem = 528/20 nm). All the LUVs used in these
experiments have been stored at 4°C and were stable for at least one week.
and contained
a
binary mixture of DOPC and DOPG
(Dioleoylphosphatidylcholine DOPC, Dioleoylphosphatidylglycerol DOPG;
all >99% purity; Aventi Polar lipids, Alabaster, AL) solubilized on
chloroform/methanol solvent (2/1).
The ellipsometric angle (∆, °) and the surface pressure (Π, mN/m) were
recorded simultaneously during the kinetic absorption. Ellipsometric
Emission fluorescence spectra measurements
Emission fluorescence spectra were recorded in the range λem = [350 –
600 nm] with a Tecan infinite M200 microplate reader. An excitation
wavelength of 285 nm was chosen to record emission fluorescence
spectra of the various compounds first solubilized in DMSO, and then
diluted in buffer B (50 mM HEPES, 107 mM NaCl, 1 mM EDTA, pH 7.4) to
reach a final concentration of 10 or 25 µM in a final volume of 200 μL. To
study interactions of molecules with biomimetic membranes, in parallel, by
keeping unchanged the final volume, spectra were also recorded in
additional presence of 2 µL of LUVs or chol-LUVs previously prepared.
measurements were carried out with
a home-made automated
ellipsometer in a “null ellipsometer” configuration.[60] He–Ne laser beam (λ
= 632.8 nm, Melles Griot, France) was polarised with a Glan-Thompson
polarizer and was reflected on the surface of the trough with an incidence
angle of 52.12°. After reflection on the liquid surface, the laser light passed
through a λ/4 retardation plate, a Glan-Thompson analyser and a
photomultiplier. The analyser angle, multiplied by two, yielded the value of
the ellipsometric angle (Δ) i.e., the phase difference between parallel and
perpendicular polarisation of the reflected light. The laser beam probed a
surface of 1 mm2 and a depth in the order of 1 µm and gives insight on the
amount of interfacial matter. Indeed, this ellipsometric angle is dependent
on two parameters: the reflective index and the thickness of the interface.
Values of Δ were recorded every 4 s with a precision of ± 0.5°.
Carboxyfluorescein efflux
CF release assay has been performed in a final volume of 200 μL, using 2
μL of the CF-loaded LUVs stock solution in buffer B. Compounds to test,
solubilized in DMSO, have been then added to the solution to reach a final
concentration of 10 or 25 µM. The fluorescence has been recorded
immediately (t0) and after 4h, using a Synergy 2 microplate reader
controlled by Gen 5 software (Biotek, Colmar, France) microplate reader
at λex 485/20 nm and λem 528/20 nm, at room temperature. The
fluorescence intensities at each time point have been normalized to the
signal when CF-loaded LUVs have been disintegrated by the addition of
100 µL of a 10% (V/V) Triton X-100 solution. The percentage of CF
leakage has been calculated using the following equation:
The surface pressure was measured following a Wilhelmy method using a
filter paper as plate (Whatman, Velizy-Villacoublay, France) connected to
a microelectronic feedback system (Nima PS4, Manchester, England).
The surface pressure (π) was recorded every 4 s with a precision of ±0.2
mN/m until the equilibrium surface pressure. The surface pressure gives
insight on the lateral interaction between the interfacial film molecules.[61,62]
I −I
t
0
CF release (%) =
x 100
−I
0
[3]
I
T
Atomic Force Microscopy (AFM) observations
At the end of the kinetics (i.e. surface pressure and ellipsometric angle
stable), a Langmuir-Blodgett (LB) transfer was performed onto freshly
cleaved mica plates at constant surface pressure (the final one) by
vertically raising (1 mm/min) the mica plate through the air/liquid interface
to obtain a sample of the interfacial monolayer. AFM imaging of LB films
was performed in contact mode using a Multimode AFM (Nano-Bruker,
Palaiseau, France) under ambient conditions with scanning areas 40 µm
x 40 µm. Topographic images were acquired using silicon nitride tips on
integral cantilevers with a nominal spring constant of 60 mN/m (Bruker, CA,
USA). The forces were controlled and minimized along the imaging
process. Three different areas were imaged for each sample to assure that
the images here shown are representative for the whole sample.
where It corresponds to the fluorescence intensity after 4h of incubation of
LUVs with the studied compound, I0 is the initial fluorescence intensity of
the negative control, IT is the fluorescence intensity after CF-loaded LUVs
disruption by the addition of 10 % Triton X-100 solution. Each experiment
has been performed in triplicate.
DPH and TMA-DPH fluorescence quenching measurements
With the DPH-loaded or TMA-DPH-loaded LUVs samples prepared as
described above, fluorescence intensity was measured at room
temperature, at time 0 and 4 hours, using a Synergy 2 Biotek microplate
reader (Biotek, Colmar, France) set at 360 nm for excitation and 460 nm
for emission, with a 40 nm bandwidth. Assays were performed in a final
volume of 200 μL in black microplates, using 2 μL of the probe-loaded
LUVs stock solution in buffer B and compounds to test, solubilized in
DMSO, and added to the samples to reach a final concentration of 10 or
25 µM. Assays were performed in duplicate.
Fluorescence quenching was calculated as follows:
I
−I
probe−loaded LUV unloaded LUV
I0/I =
[4]
I
−I
probe−loaded LUV+oligopyridine unloaded LUV+oligopyridine
15
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900542
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FULL PAPER
[11] G. Burzicki, A. S. Voisin-Chiret, J. S. Oliveira Santos, S. Rault,
Tetrahedron 2009, 65, 5413–5417.
Acknowledgements
[12] G. Burzicki, A. S. Voisin-Chiret, J. S. O. Santos, S. Rault, Synthesis 2010,
2804–2810.
We are grateful for funding support from the “Ligue Contre le
Cancer” (National and Calvados committees). Dr Domenico
Iacopetta was recipient of grant from Commissione Europea,
Fondo Sociale Europeo (FSE 2007/2013-PROGRAMMA ARUE)
and Regione Calabria. This work was also financially supported
by the Region Normandie and the European Union via the
European Regional Development Fund (FEDER). Lipoid S100
and Lipoid PG18:1 were kindly provided thanks to Jean-
Guillaume Giraudon (Lipoid GmbH). The authors want to thank
Siham Hedir, and Dr Laurent Poulain (Normandie Univ, UniCaen,
Inserm U1086 Anticipe, Axe BioTICLA, Caen, France) for helpful
discussions. We acknowledge also Peggy Suzanne (Chemical
library, CERMN and SF4206 Icore, Normandie Univ, UNICAEN)
for her helpful technical support.
[13] S. Perato, A. S. Voisin-Chiret, J. Sopková-de Oliveira Santos, R. Legay,
H. Oulyadi, S. Rault, Tetrahedron 2012, 68, 1910–1917.
[14] A. S. Voisin-Chiret, M. Muraglia, G. Burzicki, S. Perato, F. Corbo, J.
Sopková-de Oliveira Santos, C. Franchini, S. Rault, Tetrahedron 2010,
66, 8000–8005.
[15] M. De Giorgi, A. S. Voisin-Chiret, J. Sopková-de Oliveira Santos, F.
Corbo, C. Franchini, S. Rault, Tetrahedron 2011, 67, 6145–6154.
[16] S. Perato, M. De Giorgi, G. Burzicki, F. Legalite, S. Rault, A. S. Voisin-
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Table of Contents
The experimental data assessed on Pyridoclax, a promising anticancer drug, and on its 18 sensibly selected structural analogs, have
permitted to assess relative differences in terms of physico-chemical properties which could be determinant for their druggability, and
hence for their drug development.
18
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