Vectorization efforts to increase Gram-negative intracellular drug concentration: A case study on HldE-K inhibitors

Article abstract of DOI:10.1021/jm400097h

In this paper, we present different strategies to vectorize HldE kinase inhibitors with the goal to improve their Gram-negative intracellular concentration. Syntheses and biological effects of siderophoric, aminoglycosidic, amphoteric, and polycationic vectors are discussed. While siderophoric and amphoteric vectorization efforts proved to be disappointing in this series, aminoglycosidic and polycationic vectors were able for the first time to achieve synergistic effects of our inhibitors with erythromycin. Although these effects proved to be nonspecific, this study provides information about the required stereoelectronic arrangement of the polycationic amines and their basicity requirements to fulfill outer membrane destabilization resulting in better erythromycin synergies.

Full text of DOI:10.1021/jm400097h

Article
pubs.acs.org/jmc
Vectorization Efforts To Increase Gram-Negative Intracellular Drug
Concentration: A Case Study on HldE‑K Inhibitors
Dmytro Atamanyuk,† Fabien Faivre,† Mayalen Oxoby,† Benoit Ledoussal,† Elodie Drocourt,‡
Franco
̧
is Moreau,‡ and Vincent Gerusz*,†
†Medicinal Chemistry and Biology, Mutabilis, 102 Avenue Gaston Roussel, 93230 Romainville, France
‡
ABSTRACT: In this paper, we present different strategies to vectorize HldE
kinase inhibitors with the goal to improve their Gram-negative intracellular
concentration. Syntheses and biological effects of siderophoric, amino-
glycosidic, amphoteric, and polycationic vectors are discussed. While
siderophoric and amphoteric vectorization efforts proved to be disappointing
in this series, aminoglycosidic and polycationic vectors were able for the first
time to achieve synergistic effects of our inhibitors with erythromycin.
Although these effects proved to be nonspecific, this study provides
information about the required stereoelectronic arrangement of the
polycationic amines and their basicity requirements to fulfill outer membrane
destabilization resulting in better erythromycin synergies.
Several vector-mediated drug delivery systems are known and
INTRODUCTION
■
may be classified depending on their mode of uptake in
bacteria. Among them, siderophoric vectorization aims at
attaching a siderophore-like functionality to the drug to take
advantage of the bacterial iron-uptake mechanism, a strategy
sometimes referred to as the “Trojan Horse” approach.12−14
With a siderophoric moiety bound to their benzothiazolyl
group, compounds 1−8 in Table 1 illustrate this strategy.
By tethering an aminoglycoside moiety to the drug,
aminoglycosidic vectorization targets the OM passage via
porins for the smaller-size aminoglycosides and/or by self-
promoted pathway via displacement of the stabilizing dications
of the LPS.15 Then in a strategy reminiscent of the “Trojan
Horse” approach, the IM would be crossed by an energy-
dependent aminoglycoside uptake.16 The aminoglycoside
kanamycin was attached to the benzothiazole part of our series
via a triazole linker to illustrate this strategy (compound 9,
Table 2).
Amphoteric antibacterials such as some fluoroquinolones or
penicillins penetrate the OM via passive diffusion17 through the
porins in their zwitterionic forms,18,19 a process facilitated in
some cases by formation of a Mg2+ chelate.20 Passage though
the IM for amphoteric fluoroquinolones or tetracyclines is
subsequently achieved by diffusion of the equilibrated neutral
microspecies.20,21 Therefore, amphoteric vectorization, consist-
ing of installing acidic and/or basic functional groups to an
inhibitor to make it amphoteric, could lead to better OM
penetration via its zwitterionic form and better IM influx via its
uncharged neutral form. To illustrate this strategy, compounds
10 and 11 in Table 3 were designed with a basic amine and an
Rates of multidrug-resistant Gram-negative organisms are
increasing at an alarming speed, outpacing in some cases
those of methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant enterococci (VRE).1 This, combined with
the shortage of new drugs in the antibacterial development
pipeline,2 constitutes a major health concern.
One approach to address this issue is to identify inhibitors
with novel modes of action based on the new targets
originating from the rise of bacterial genomics in the 1990s.3
Unfortunately, the general outcome has proven disappointing
so far,4 some authors even cautioning against the exploitation of
cytosolic targets with regard to the difficulty of reaching the
required active concentration in the bacterial cell.5 Indeed in
Gram-negative bacteria, obtaining the active intracellular drug
concentration with reversible synthetic inhibitors represents a
formidable challenge, since the compound must first cross a
hydrophilic outer membrane (OM)6 followed by a lipophilic
inner one (IM) and finally resist a battery of efflux
mechanisms.7−9
We have previously reported10 the nanomolar optimization
of 1,2,4-triazine-based inhibitors of HldE-kinase, a novel
antivirulence target involved in the synthesis of the inner
core of lipopolysaccharides (LPS).11 Some of these compounds
demonstrate specific LPS biosynthetic inhibition on efflux-
deleted Gram-negative strains, yet are inactive at relevant
pharmacological doses on wild pathogens because of poor
membrane permeation and/or efficient efflux. A potential
method to circumvent these problems is the adjunction of
functional groups or covalently attached molecules called
vectors to the inhibitor in the hope of increasing intracellular
drug concentration by improving membrane permeation and/
or reducing efflux (Figure 1).
Received: October 18, 2012
Published: February 27, 2013
© 2013 American Chemical Society
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Figure 1. Schematic representation of the mechanisms affecting Gram-negative drug intracellular concentration.
acidic phenolic group to allow them to exist partly in both
zwitterionic and neutral forms.
Sulfonylation of amino compound 2710 provided derivative
28 that was demethylated to afford compound 3, whereas its
amidation using standard coupling reagents provided com-
pound 8 as illustrated in Scheme 3. Compound 5 was obtained
in a similar way by coupling 27 with comenic acid.
The self-promoted uptake involves the displacement of LPS-
cross-bridging cations by polycationic compounds, allowing
their entry into the periplasm after LPS destabilization.15,22,23
Polycationic vectorization is the process of adding cationic
functional groups to a drug in order to increase its OM
permeation. In some instances, such chemical modifications
have been shown to broaden the in vitro activity spectrum of
glycopeptides against Gram-negative pathogens.24,25 To illus-
trate this strategy, compounds 12−18 in Table 4 have been
appended with polyamine side chains.
Hydroxy compound 2910 was derivatized as illustrated in
Scheme 4 by mesylation followed by substitution using a
thionucleophile to provide compound 7 or using amino-
nucleophile 3228 to afford after Boc deprotection compound
17. The low reported yield of this deprotection might be
explained by purification over a strong cation exchange column
(SCX), which may have partially retained the product.
Derivative 13 was obtained in a similar way using 1-
methylpiperazine as nucleophile.
Encouraged by SAR data10 and crystallographic results26
indicating space available at both extremities of our HldE-K
inhibitors, we embarked on a vectorization program using these
strategies. In the present report, we describe the syntheses,
biological results, and the effects of these vectors on Gram-
negative intracellular concentration of our inhibitors.
Propargylamide derivative 34, prepared from acid 1910 using
similar conditions as described in Scheme 1, was coupled to N-
protected 6″-azido-kanamycin A 3329 under copper-catalyzed
Huisgen conditions to afford compound 35. This cycloaddition
was run according to the mentioned reference, which did not
use any nitrogen-type ligands to accelerate the reaction. Since
intermediate 35 was prepared in a sufficient amount for the
final deprotection and biological evaluation, the reported yield
of 12% was not further optimized. Final Boc removal under
acidic conditions provided compound 9 as illustrated in
Scheme 5.
Phenol derivative 10 was obtained by demethylation of the
corresponding methyl ether analogue 3610 using Lewis acidic
conditions as illustrated in Scheme 6. Compound 11 was
prepared following the same procedure from the amido
analogue 37.10
CHEMISTRY
■
The vectorization of our reported HldE-K inhibitors10 was
carried out using concise and straightforward synthetic routes.
Acidic compound 1910 was derivatized using standard coupling
reagents in amides or esters as illustrated in Scheme 1. Reaction
with hydroxylamine provided the hydroxamic acid derivative 1.
Esterification with alcohol 2027 led to ester 21, which was
deprotected under acidic conditions to afford compound 6,
while amidification with 1,4-bis-Boc-1,4,7-triazaheptane led to
amide 22, which was deprotected under acidic conditions to
afford compound 14. Similar conditions were used to
synthesize target amides 15, 16, and 18.
Aminoethyl derivative 12 was prepared via nucleophilic
substitution of chlorotriazine 3810 by amine 2410 followed by
Boc removal, primary amine protection, aniline substitution
with 2-chloro-N,N-dimethylethylamine, and final deprotection
as illustrated in Scheme 7. The 13% reported yield for the final
Compound 2 was accessed by nucleophilic substitution of
chlorotriazine 2310 by aminomethylthiazole 2410 followed by
Boc and THP deprotection and final carbamoylation as
illustrated in Scheme 2.
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Table 1. Siderophoric Vectorization: Comparison of Vectorized and Unvectorized Compounds
a
b
Synergistic MIC in the presence of 1 μg/mL erythromycin. Synergistic MIC in the presence of 0.1 μg/mL erythromycin in iron depleted medium.
c
N/A: not applicable. Synergistic MIC in the presence of 8 μg/mL erythromycin.
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Table 2. Aminoglycosidic Vectorization: Comparison of Vectorized and Unvectorized Compounds
a
b
Synergistic MIC in the presence of 1 μg/mL erythromycin. Synergistic MIC in the presence of 8 μg/mL erythromycin.
Table 3. Amphoteric Vectorization: Comparison of Vectorized and Unvectorized Compounds
a
b
c
Synergistic MIC in the presence of 1 μg/mL erythromycin. Synergistic MIC in the presence of 8 μg/mL erythromycin. Species distribution based
d
e
f
on predicted pKa microconstants (ACDLabs, version 12). Zwitterionic form. Neutral form (uncharged). Not applicable.
one-pot two steps of this procedure might be explained by the
low nucleophilic reactivity of the aniline-type nitrogen in the
first-step nucleophilic substitution, which could be further
deactivated by the electron-deficient triazine.
expressed as a synergistic MIC of inhibitor in association
with erythromycin at its own MIC/4 concentration against E.
coli K12 MG1655 Δ-acrAB-Δtolc, E. coli K1 (018:K1:H7) wild
type, and Δtolc.10,30 In both mutant strains, deletion of the
major efflux pump AcrAB/TolC (in part or entirely) leads to an
increased susceptibility toward amphiphilic and lipophilic
antibiotics.31 In the most promising synergy cases, bacterial
LPS electrophoresis experiments10 were carried out with the
inhibitor alone to determine its capacity to specifically inhibit
RESULTS AND DISCUSSION
■
The biological activities of the compounds were determined by
measuring their enzymatic inhibitory concentrations on E. coli
HldE-K expressed as IC50, as well as their ability to synergize
the Gram-negative antibacterial potential of erythromycin
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Table 4. Polycationic Vectorization: Comparison of Vectorized and Unvectorized Compounds
a
b
c
Synergistic MIC in the presence of 1 μg/mL erythromycin. Synergistic MIC in the presence of 8 μg/mL erythromycin. Species distribution based
d
e
f
g
on predicted pKa microconstants (ACDLabs, version 12). Neutral form (uncharged). Monocationic form. Dicationic form. Along with 2% of the
tricationic form.
full LPS biosynthesis and therefore provide a marker for its
intracellular concentration.
Since hydroxamate-type siderophores are known to be
recognized by E. coli FhuD binding protein,32 the carboxylic
acid moiety of 19 was transformed into an iron-chelating
hydroxamic acid group in compound 1 with the hope of
benefiting from this active transport system (Table 1). While
enzymatic potency was preserved, a 16-fold gain in synergy
with erythromycin was achieved on E. coli K12 Δ-acrAB, Δ-
tolC, an efflux-deleted strain. Repeating the synergy experiment
under iron-depleted conditions to maximize the expression of
bacterial siderophoric influx did not lead to any significant
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a
Scheme 1. Derivatization of Compound 19
a
Reagents: (a) hydroxylamine, DIPEA, EDCI, HOBt, DMF, rt (37%); (b) EDCI, DMAP, THF, rt (32%); (c) TFA, DCM, rt (46%); (d) 1,4-bis-
Boc-1,4,7-triazaheptane, EDCI, HOBt, DIPEA, DMF, rt (56%); (e) HCl, dioxane, methanol, DCM, rt (63%).
a
Scheme 2. Synthesis of Compound 2
a
Reagents: (a) DIPEA, ACN, 85 °C (45%); (b) HCl, dioxane, 0 °C to rt (62%); (c) 4-ethyl-2,3-dioxo-1-piperazinecarbonyl chloride, DIPEA, THF,
0 °C to rt (29%).
a
Scheme 3. Derivatization of Compound 27
a
Reagents: (a) 3,4-dimethoxybenzenesulfonyl chloride, DIPEA, DCM, 0 °C to rt (78%); (b) BBr3, DCM, −78 to −30 °C (20%); (c) (R)-2-(2-
hydroxyphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid, EDCI, DMAP, DIPEA, HOBt, rt (30%).
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a
Scheme 4. Derivatization of Compound 29
a
Reagents: (a) MsCl, TEA, THF, 0 °C to rt (90%); (b) 6-hydroxy-3-mercapto-2-methyl-2H-[1,2,4]triazin-5-one, TEA, DMF, 60 °C (28%); (c) [3-
(3-tert-butoxycarbonylaminopropylamino)propyl]carbamic acid tert-butyl ester 32, ACN, 85 °C (43%); (d) HCl, dioxane, DCM, 0 °C to rt (23%).
a
Scheme 5. Synthesis of Kanamycin Derivative 9
a
Reagents: (a) CuSO4, sodium ascorbate, tert-BuOH, water, rt (12%); (b) HCl, dioxane, MeOH, rt (64%).
a
Scheme 6. Synthesis of Compound 10
a
Reagents: (a) BBr3, DCM, −78 °C (44%).
a
Scheme 7. Synthesis of Compound 12
a
Reagents: (a) DIPEA, ACN, 85 °C (58%); (b) (i) HCl (aqueous 37%), 0 °C; (ii) (Boc)2O, TEA, THF, rt (73%); (c) (i) 2-chloro-N,N-
dimethylethylamine hydrochloride, NaHCO3, NaI, EtOH, 80 °C; (ii) HCl, dioxane, MeOH, rt (13%).
improvement. This cast some doubts of whether the improve-
ment of erythromycin synergy seen with 1 vs 19 was truly due
to siderophoric uptake followed by specific cytosolic inhibition
of HldE-K. In this case, the observed synergy might possibly be
explained by the hydroxamate-induced displacement of LPS-
stabilizing cations. Adding other iron-chelating moieties to the
right part of our unvectorized inhibitors such as catechol (3),
acetylsulfamoyl (4), 1,5-dihydroxy-4-pyridone (6), and 6-
hydroxy-2-methyl-2H-[1,2,4]triazin-5-one (7) led to similar
results. Enzymatic activity could be maintained in the low
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nanomolar range, and in some instances potent synergistic
activity was obtained on E. coli K12 Δ-acrAB, Δ-tolC (2 μg/mL
for 7 vs 32 μg/mL for 19 with comparable enzymatic activities).
Yet again, iron-depleted conditions could not significantly
improve these synergies. Although some of these vectors have
been able to achieve good periplasmic accumulation through
demonstrated siderophoric influx,33 they could not lead in our
series to enhanced cytosolic concentrations under iron-depleted
conditions nor on wild-type E. coli K1 strain. Tethering in
compound 8 the 2-(2-hydroxy-phenyl)-4,5-dihydrothiazolyl
moiety commonly found in Gram negative siderophores such
as yersiniabactin or pyochelin led to a decrease in enzymatic
activity and a complete loss of synergistic activity on our
bacterial strains. Adding to compound 39 the piperazinedione
vector found in piperacillin also led to a decrease in enzymatic
activity (520 nM in 2 vs 69 nM in 39). However, for this
compound the synergistic bacterial activity on E. coli K12 Δ-
acrAB, Δ-tolC could be significantly improved under iron-
depleted conditions, indicating a possible true siderophoric
uptake. A similar significant improvement of synergistic activity
under iron-depleted conditions was observed for 5, which is
vectorized with a kojic acid derivative known for its iron-
chelating properties.34,35 Compound 5 also presents an
interesting enzymatic potency below 100 nM. These two
promising compounds unfortunately were found to be inactive
on wild E. coli K1 strain, even under iron-depleted conditions.
These results indicate that our vectors based on the
siderophoric strategy, although displaying in some instances
improvements on efflux-deleted strains, are not potent enough
to enhance the cytosolic concentration of our compounds in
wild strains. This may suggest the need for a more complete
siderophoric moiety or some drug-release mechanism in the
periplasm to better cross the IM.36
Aminoglycosidic vectorization was then attempted by
tethering the aminoglycoside kanamycin to a representative
member of our series. The resulting compound 9 (Table 2) still
displayed potent enzymatic activity despite the added steric
bulk. More interestingly, synergy with erythromycin was
achieved for the first time on E. coli K1 wild strain (compare
9 and 40). Control experiments were run to assess that the
kanamycin moiety did not impart any antibacterial activity in 9
at these doses.30 Control checkerboard assays were also run to
rule out any potential synergies between the unvectorized
compound 41 and erythromycin or between the vector itself,
kanamycin, and erythromycin.37 On E. coli K1 wild strain, the
MIC of erythromycin remained at 16 μg/mL independent of
the concentration of 41 (0−32 μg/mL). On the same strain,
kanamycin alone displayed a MIC of 8 μg/mL, while it
demonstrated a pure additive effect in combination with
erythromycin. In view of this promising result, an LPS
electrophoresis assay was then run to determine the ability of
9 to inhibit LPS biosynthesis on this strain. However, at 30 μg/
mL, no inhibition of LPS biosynthesis was observed, suggesting
that the synergy with erythromycin is not specific. One could
speculate that this effect is probably induced by the amphiphilic
character of 9, which may facilitate the permeation of
erythromycin.
enhance their charge distribution, an acidic phenolic hydroxyl
was positioned to the right part of 36, while the basic amine left
part was conserved. The resulting compound (10, Table 3)
presents 13.3% of zwitterionic form at the bacterial assay pH.
Despite a 10-fold enzymatic potency loss, 10 displayed a
slightly better synergistic activity with erythromycin on E. coli
K12 Δ-acrAB, Δ-tolC in comparison to the nonzwitterionic
analogue 36. However, this compound proved to be inactive on
E. coli K1 wild strain. Concerned that the low amount of
uncharged neutral form of 10 (1.5% at pH 7.4, Table 3) might
be a limitation to cross the IM, compound 11 was designed
with a higher percentage of neutral form (44.3%) while still
harboring a significant amount of zwitterionic form (6.6%).
Here again despite a 10-fold enzymatic potency loss, 11
displayed a slightly better synergistic activity with erythromycin
on E. coli K12 Δ-acrAB, Δ-tolC in comparison to the
nonzwitterionic analogue 36 but was still inactive on the wild
strain. The results of amphoteric vectorization therefore proved
to be disappointing in our series because of the enzymatic
activity loss imparted by this strategy, preventing it from
expressing its real potential. Another factor may also be the
inadequate geometry of our charge-bearing groups, since
stereoelectronic complementarity between the compound and
the channel should be present for proper translocation.41
Adjunction of cationic groups to our representative inhibitors
was then attempted with the hope of penetrating the bacterial
cell via membrane destabilization. Initial trials with protonable
amines at both sides of our compounds resulted in a marked
decrease of enzymatic activity and an absence of synergy with
erythromycin (compound 12, Table 4). The presence of 85%
of the dicationic form of 12 was not able to permeabilize the
antibiotic in the bacteria, suggesting that another stereo-
electronic arrangement of the charges, possibly closer to each
other or in a more “surfactant-like” arrangement, is required to
achieve membrane destabilization. To reach this goal, the
charges were positioned to the right side of our inhibitors. In
compound 13, the piperazine nitrogens mutually decrease their
pKa by inductive effect and no dicationic form is predicted at
pH 7.4. Despite displaying potent enzymatic activity, this amine
disposition did not enhance the synergy with erythromycin in
comparison to the unvectorized analogue 41. Increasing the
basicity of the protonable amines (14, 15) and their numbers
(16, 17) resulted in an increase of the dicationic form,
culminating with the presence of a small amount of the
tricationic form in 17. This was directly correlated with a
marked improvement of the synergistic activity with eryth-
romycin, even on the wild strain. These data also indicate that
some flexibility in the amine disposition is tolerated as long as a
high level of the dicationic form is present. Here again, a
control checkerboard assay was run to rule out any potential
synergies between the polycationic vector of compound 17 and
erythromycin.42 On E. coli K1 wild strain, the MIC of
erythromycin remained at 16 μg/mL independent of the
concentration of bis(3-aminopropyl)amine (0−64 μg/mL).
Since enzymatic potency was still submicromolar for all these
compounds, LPS electrophoresis assays were run to determine
their ability to inhibit LPS formation in E. coli K1 Δ-tolC strain.
For the difference of 41, which displays almost complete LPS
biosynthetic inhibition at 32 μg/mL, none of these polycationic
compounds were able to inhibit LPS formation at this dose.
This suggests that these compounds do not reach their active
cytosolic concentration but are still capable of destabilizing the
OM to allow translocation of erythromycin in E. coli in a
Enterobacterial porins such as OmpF display a hydrophilic
channel with negative residues facing the opposite half-ring of
positively charged ones.38,39 This channel constitutes a
stereoelectronic filter selecting small polar molecules, favoring
in some instances the translocation of zwitterionic com-
pounds.40 To increase the polarity of our inhibitors and
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are in fact more permeable to hydrophobic antibiotics because of their
reduced LPS hydrophilic barrier; this is the basic principle of this
simple assay. All chemicals were from Sigma unless otherwise stated.
From early to log phase preculture, the bacterial suspension was
prepared in ca-MHB (Becton-Dickinson) or M9 supplemented (M9S)
medium to obtain a final inoculum of 5 × 105 CFU/mL. M9S was
made of M9 medium supplemented with D-glucose (100 g/L),
thiamine (10 g/L), casein (20 g/L), and apotransferrin (10 g/L).
Strains were E. coli K12 MG1655 Δ-acrAB-Δtolc, E. coli K1
(018:K1:H7) wild type, and Δtolc. Growth conditions included
erythromycin, or not, at respective concentrations of 1, 8, 1 μg/mL in
ca-MHB and 0.13, 2, 0.25 μg/mL in M9S. The MIC of studied
compounds were determined against these strains and conditions by
visual inspection in polystyrene 96-well microplates containing serial
dilutions of compounds in 2% DMSO in a final volume of 100 μL and
after overnight incubation at 35 °C.
Checkerboard Assays. The effect of combining two compounds
against E. coli K1 (018:K1:H7) was studied by checkerboard assay.
From early to log phase preculture, the bacterial suspension was
prepared in ca-MHB (Becton-Dickinson) to obtain a final inoculum of
5 × 105 CFU/mL. The combined serial dilutions contained kanamycin
(Sigma, ref K4000) or compound 41 or bis(3-aminopropyl)amine
(Aldrich ref I1006) with erythromycin (Fluka, ref 45674). The MICs
of studied combinations were determined by visual inspection in
polystyrene 96-well microplates in a final volume of 100 μL and 2%
DMSO after overnight incubation at 35 °C.
Inhibition of E. coli K1 Wild Type or Δtolc LPS Biosynthesis.
Principle. E. coli K1 (018:K1:H7) is a newborm meningitidis E. coli
(NMEC) strain that displays a typical LPS made of lipid A successively
branched with the inner and outer core oligosaccharides and finally
with the O-antigen repeats. The inner core contains several heptose
residues. An inhibitor of the LPS heptosylation pathway should
therefore reduce dramatically the size of LPS from full-length to the
so-called “Re-LPS” limited to lipid A branched with two Kdo residues.
A simple way of monitoring LPS size and composition consists of
running LPS gel electrophoresis: a wild type E. coli strain displays
several bands including those for full and core LPS but none for Re-
LPS. On the contrary, a Δ-hldE mutant defective for LPS-
heptosylation biosynthesis displays only the Re-LPS band.
Bacterial Culture. The effect of heptosylation inhibitors on E. coli
LPS was assessed as described below. The compounds to be tested
were prepared in deionized water/DMSO (50/50) solutions and
added (25 μL) to sterile culture microtubes. The strains used in this
study were E. coli K1 (018:K1:H7) wild type or Δtolc. The bacteria
were isolated on tryptic soy agar (TSA) overnight. Isolated colonies
were cultured in 10 mL of Luria−Bertani medium (LB) at 37 °C to an
optical density of typically 0.15. These exponentially growing bacteria
were finally diluted to 5 × 105 CFU/mL and added to each well (225
μL) for incubation with the compounds at 37 °C for approximately 5
h, up to an optical density of 0.2−0.4.
nonspecific manner. Here again, one could speculate that this
effect is probably induced by the amphiphilic character of the
polycationic moieties linked to the lipophilic inhibitors, which
may facilitate the permeation of erythromycin. Compound 18
was then designed with a balance of 8% of the dicationic form
to destabilize the OM along with 6% of the uncharged form
aiming at a better IM passage. The synergy results were under
par, reinforcing the observation that a high amount of dicationic
form is required for erythromycin synergy in this polycationic
vectorization strategy.
CONCLUSION
■
We have described our efforts at vectorizing nanomolar
inhibitors of HldE-kinase, a Gram-negative cytosolic target,
using different strategies. Siderophoric vectorization led in a few
cases to some improvement on efflux-pump deleted bacteria,
possibly more related to the displacement of LPS-stabilizing
cations than true siderophoric uptake. However, this strategy
did not prove to be effective in wild bacteria, suggesting the
need for a more complete siderophoric vector or some drug-
release mechanism in the periplasm to better cross the IM.
Aminoglycosidic vectorization afforded for the first time
erythromycin synergy with our inhibitors in wild strains, and
yet it was a nonspecific effect probably induced by the
aminoglycoside displacement of the stabilizing dications of the
LPS. Amphoteric vectorization proved to be disappointing in
our examples because of the resulting loss of enzymatic activity
that prevented an assessment of the real potential of this
approach. Polycationic vectorization generated in quite a few
instances erythromycin synergies with our inhibitors in wild
bacteria. Like in the case of aminoglycosidic vectorization, this
was achieved in a nonspecific manner probably induced by OM
destabilization while the positive charges associated with the
vector were unable to cross or destabilize the IM. Nevertheless,
these polycationic examples provide interesting considerations
about the required stereoelectronic arrangement of the amines
and their basicity requirements to fulfill OM destabilization
resulting in better erythromycin synergies. This work highlights
the continuing challenge of finding suitable vectors to increase
Gram-negative intracellular drug concentration.
EXPERIMENTAL SECTION
■
HldE-K Enzyme Assay. The assay buffer “AB” contained 50 mM
Hepes, pH 7.5, 1 mM MnCl2, 25 mM KCl, 0.012% Triton-X100, 1
mM dithiothreitol (DTT), and 0.1 μM myelin basic protein (MBP).
The following components were added to a white polystyrene Costar
plate to a final volume of 30 μL: 10 μL of inhibitor dissolved in
DMSO/water, 50/50, and 20 μL of HldE of E. coli in AB. After 30 min
of preincubation at room temperature, an amount of 30 μL of
substrates mix in AB was added to each well to a final volume of 60 μL.
This reaction mixture was then composed of 3 nM HldE, 0.2 μM β-
heptose 7-phosphate (custom synthesis), and 0.2 μM ATP (Sigma) in
assay buffer. After 30 min of incubation at room temperature, an
amount of 200 μL of the mix was added to a final volume of 260 μL,
including the following constituents at the respective final concen-
trations: 5000 light units/mL luciferase (Sigma), 30 μM D-luciferin
(Sigma), 100 μM N-acetylcysteamine (Aldrich). Luminescence
intensity was immediately measured on Luminoskan (Thermofischer)
and converted into inhibition percentages. For IC50 determinations,
the inhibitor was tested at 6−10 different concentrations, and the
related inhibitions were fitted to a classical Langmuir equilibrium
model using XLFIT (IDBS).
LPS Extraction. Bacterial cultures were normalized via OD
determination, pelleted, and washed with 1 mL of phosphate buffered
saline (PBS). The pellets were then denatured for 10 min at 95−100
°C in 50 μL of sodium dodecyl sulfate 0.2% (SDS), β-mercaptoethanol
1%, glycerol 36%, Tris, pH 7.4, 30 mM, and bromophenol blue
0.001%. Samples were cooled to room temperature, supplemented
with 1.5 μL of proteinase K at 20 mg/mL, incubated for 1 h at 55 °C,
and centrifuged for 30 min at 13 000 rpm at 25 °C. The resulting
supernatant containing LPS was finally analyzed by SDS−PAGE
electrophoresis.
LPS SDS−PAGE Electrophoresis. Polyacrylamide gels (16% and 4%
acrylamide for separation and concentration, respectively) were
prepared, loaded with 8 μL of LPS extracts, and migrated.
Silver Staining. Gels were incubated overnight in 5% acetic acid/
40% ethanol/deionized water, treated with 1% periodic acid/5% acetic
acid for 15 min, washed 4 times for 10 min in deionized water, and
finally incubated for 18 min in the dark in a silver nitrate solution
composed of 56 mL of NaOH 0.1 N, 4 mL of ammoniac 33%, 45 mL
of AgNO3 5% (Tsai and Frasch), and 195 mL of deionized water. Gels
were then washed extensively in deionized water for 30 min and
Antimicrobial Synergy with Erythromycin. Erythromycin is
much more potent on E. coli Δ-hldE (MIC= 0.5−2 μg/mL) than on E.
coli wild type strains (MIC = 32−64 μg/mL). The hldE-deleted strains
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incubated for 10−15 min (up to LPS bands apparition) in the mixture
composed of 300 mL of deionized water, 300 μL of formaldehyde
36.5% (Fluka), and 100 μL of citric acid 2.3 M. The reaction was
stopped by incubating the gels in acetic acid 10% for 5 min. Gels were
finally washed in deionized water, numerized with a Samsung PL51
camera, and analyzed by ImageJ software. The percentage of inhibition
of LPS heptosylation was defined as the relative area of the Re-LPS
band compared to the cumulated areas of Re-LPS and Core-LPS
bands.
filtered, washed with MeOH (2 mL) and water (0.5 mL), then dried to
afford 6 (9.2 mg, 46%) as a yellow powder. MS (ESI+): 601, 603 [M +
H]+. 1H MNR (400 MHz, DMSO-d6) δ: 9.15 (1H, br s); 8.86 (1H, s);
7.94 (1H, d, J = 8.7 Hz); 7.75 (1H, s); 7.70−7.52 (4H, m); 7.11 (1H,
d, J = 8.7 Hz); 6.81 (1H, s); 5.19 (2H, s); 5.12−4.96 (4H, m).
(2-tert-Butoxycarbonylaminoethyl)-{2-[2-(2-{[5-(2,6-
dichlorophenyl)[1,2,4]triazin-3-ylamino]methyl}benzothiazol-
5-yloxy)acetylamino]ethyl}carbamic Acid tert-Butyl Ester (22).
To acid 1910 (40 mg, 0.060 mmol) were added 1-hydroxybenzotriazol
(30 mg, 0.222 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide hydrochloride (50 mg, 0.260 mmol), and dry dimethylforma-
mide (0.5 mL). The mixture was stirred for 1 h at room temperature.
Then to the mixture were added diisopropylethylamine (33 μL, 0.189
mmol) and 1,4-bis-Boc-1,4,7-triazaheptane (29 mg, 0.095 mmol). The
mixture was stirred overnight and then partitioned between ice−water
and ethyl acetate. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by preparative TLC on silica
gel (10% of methanol in dichloromethane) to afford 22 (36 mg, 56%)
as a colorless oil. MS (ESI+): 747−749 [M + H]+.
General Experimental. All reactions were carried out under inert
(nitrogen or argon) atmosphere unless indicated otherwise. Reagents
and solvents were obtained from commercial sources and were used
without further purification. Celite is a filter aid composed of
diatomaceous silica and is a registered trademark of Celite
Corporation. Analtech silica gel GF and E. Merck silica gel 60 F-254
thin layer plates were used for thin layer chromatography. Flash
chromatography was carried out on Flashsmart Pack cartridge irregular
silica 40−60 μm or spherical silica 20−40 μm. Preparative thin layer
chromatography was carried out on Analtech silica gel GF 1000 μm 20
cm × 20 cm. Yields refer to purified products and are not optimized.
N-[2-(2-Aminoethylamino)ethyl]-2-(2-{[5-(2, 6-
dichlorophenyl)[1,2,4]triazin-3-ylamino]methyl}benzothiazol-
5-yloxy)acetamide (14). To a solution of 22 (36 mg, 0.048 mmol)
in 1.5 mL of dichloromethane−methanol (2:1) was added HCl 4 M in
dioxane (300 μL, 1.20 mmol), and the mixture was stirred for 3 h at
room temperature. The mixture was evaporated to dryness to afford
14 as a brown solid hydrochloride salt (34 mg, 63%). MS (ESI+): 547
1
All new compounds gave satisfactory analytical data. H NMR spectra
were recorded at 300 or 400 MHz on a Bruker instrument, and
̈
chemical shifts are reported in parts per million (δ) downfield from the
internal standard tetramethylsilane. Mass spectra were obtained using
electrospray (ESI) ionization techniques on an Agilent 1100 series
LCMS instrument. HPLC (analytical and preparative) was performed
on an Agilent 1100 HPLC instrument with diode array detection.
Preparative HPLC was performed at 0.7 mL/min on a Thermo
Electron, Hypersil BDS C-18 column (250 mm × 4.6 mm, 5 μm)
using a gradient of acetonitrile and water with 0.1% TFA (50% in
acetonitrile to 100% and then back to 50%). The tested compounds
were determined to be >95% pure via HPLC.
1
[M + H]+. H MNR (400 MHz, DMSO-d6) δ: 9.36 (2H, br s); 8.86
(1H, s); 8.46 (1H, t, J = 5.5 Hz); 8.25 (3H, br s); 7.94 (1H, d, J = 8.8
Hz); 7.70−7.53 (4H, m); 7.17 (1H, dd, J1 = 8.7 Hz, J2 = 1.9 Hz); 5.03
(2H, br s); 4.62 (2H, s); 3.23−3.11 (6H, m).
N,N-Bis(3-aminopropyl)-2-(2-{[5-(2,6-dichlorophenyl)[1,2,4]-
triazin-3-ylamino]methyl}benzothiazol-5-yloxy)acetamide
(15). 15 was obtained from acid 1910 and [3-(3-tert-
butoxycarbonylaminopropylamino)propyl]carbamic acid tert-butyl
ester 3228 in similar conditions as described for 22 and 14. MS
2-{[2-({[5-(2,6-Dichlorophenyl)-1,2,4-triazin-3-yl]amino}-
methyl)-1,3-benzothiazol-5-yl]oxy}-N-hydroxyacetamide (1).
To acid 1910 (55 mg, 0.119 mmol) were added 1-hydroxybenzotriazol
(321 mg, 0.155 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide hydrochloride (34 mg, 0.179 mmol), and dry dimethylforma-
mide (1 mL). The mixture was stirred for 1 h at room temperature.
Then to the mixture were added diisopropylethylamine (23 μL, 0.130
mmol) and hydroxylamine hydrochloride (9 mg, 0.130 mmol). The
mixture was stirred overnight and then partitioned between saturated
NaHCO3 and ethyl acetate. The organic layer was dried over Na2SO4
and concentrated. The residue was purified by preparative TLC on
silica gel (10% of methanol in dichloromethane) to afford 1 (21 mg,
37%). MS (ESI+): 477 [M + H]+. 1H MNR (400 MHz, DMSO-d6) δ:
10.88 (1H, s); 8.99 (1H, s); 8.87 (1H, s); 7.91 (1H, d, J = 8.6 Hz);
7.70−7.48 (4H, m); 7.09 (1H, d, J = 7.3 Hz); 5.10−4.8 (2H, m); 4.55
(2H, s).
(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]methyl}-
benzothiazol-5-yloxy)acetic Acid 1,5-Bis(4-methoxybenzy-
loxy)-4-oxo-1,4-dihydropyridin-2-ylmethyl Ester (21). To acid
1910 (50 mg, 0.11 mmol) were added 2-hydroxymethyl-1,5-bis(4-
methoxybenzyloxy)-1H-pyridin-4-one 2027 (44 mg, 0.11 mmol), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (85 mg, 0.44
mmol), 4-dimethylaminopyridine (DMAP) (4 mg, 0.03 mmol), and
dry THF (2 mL). The mixture was stirred for 15 h at room
temperature, then concentrated under reduced pressure and then
partitioned between water and ethyl acetate. The organic layer was
dried over Na2SO4 and concentrated. The residue was purified by
preparative TLC on silica gel (10% of methanol in dichloromethane)
to afford compound 21 as a yellowish oil (30 mg, 32%). MS (ESI+):
841, 843 [M + H]+.
1
(ESI+): 575, 577 [M + H]+. H NMR (400 MHz, DMSO-d6) δ: 9.18
(1H, br s); 8.87 (1H, s); 8.11 (3H, m); 7.98−7.86 (4H, m), 7.72−7.53
(3H, m), 7.47 (1H, br s), 7.07 (1H, dd, J1 = 8.7 Hz, J2 = 2.1 Hz); 5.05
(2H, br s), 4.93 (2H, s), 3.44 (2H, t, J = 7.5 Hz); 3.37 (2H, t, J = 7.0
Hz); 2.86 (2H, m), 2.76 (2H, m), 1.94 (2H, quint, J = 7.3 Hz); 1.81
(2H, quint, J = 7.0 Hz).
N-{2-[Bis(2-aminoethyl)amino]ethyl}-2-(2-{[5-(2,6-
dichlorophenyl)[1,2,4]triazin-3-ylamino]methyl}benzothiazol-
5-yloxy)acetamide (16). 16 was obtained from acid 1910 and {2-[(2-
aminoethyl)-(2-tert-butoxycarbonylaminoethyl)amino]ethyl}carbamic
acid tert-butyl ester43 in similar conditions as described for 22 and 14.
MS (ESI+): 590, 592 [M + H]+. 1H NMR (400 MHz, MeOD) δ ppm:
8.73 (1H, s); 7.87 (1H, d, J = 8.9 Hz); 7.51 (4H, br s); 7.23 (1H, dd, J1
= 8.9 Hz, J2 = 2.4 Hz); 5.11 (2H, br s); 4.70 (2H, s); 3.51 (2H, m);
3.35 (1H, s); 3.19−2.80 (11H, m).
2-(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]-
methyl}benzothiazol-5-yloxy)-N-(2-dimethylamino-1-
dimethylaminomethylethyl)acetamide (18). 18 was obtained
from acid 1910 and N,N,N″,N″-tetramethylpropane-1,2,3-triamine44 in
similar conditions as described for 22 and 14. MS (ESI+): 589, 591 [M
1
+ H]+. H NMR (400 MHz, MeOD) δ ppm: 8.75 (1H, s); 7.91 (1H,
d, J = 8.9 Hz); 7.55 (4H, m); 7.21 (1H, dd, J1 = 8.9 Hz, J2 = 2.4 Hz);
5.11 (2H, br s); 4.69 (2H, s); 3.59 (1H, dd, J1 = 13.5 Hz, J2 = 5.6 Hz);
3.38 (1H, dd, J1 = 13.6 Hz, J2 = 5.6 Hz); 2.95 (1H, m); 2.68 (1H, m);
2.52 (1H, m); 2.40 (12H, s).
{2-[2-({5-[2-Methyl-6-(tetrahydropyran-2-yloxy)phenyl]-
[1,2,4]triazin-3-ylamino}methyl)benzothiazol-5-yloxy]ethyl}-
carbamic Acid tert-Butyl Ester (25). To a mixture of 3-chloro-5-[2-
methyl-6-(tetrahydropyran-2-yloxy)phenyl][1,2,4]triazine 2310 (150
mg, 0.48 mmol) in acetonitrile (2 mL) were added [2-(2-amino-
methylbenzothiazol-5-yloxy)ethyl]carbamic acid tert-butyl ester 2410
(171 mg, 0.53 mmol) and DIPEA (125 μL, 0.72 mmol), and the
mixture was stirred for 14 h at 85 °C. Reaction mixture was
concentrated to dryness and then dissolved in EtOAc. The formed
precipitate of the remaining starting compound 5 was filtered off and
discarded. The filtrate was washed with water, and the organic layers
(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]methyl}-
benzothiazol-5-yloxy)acetic Acid 1,5-Dihydroxy-4-oxo-1,4-di-
hydropyridin-2-ylmethyl Ester (6). To a solution of compound 21
(28 mg, 0.033 mmol) in dichloromethane (1 mL) was added dropwise
trifluoroacetic acid (TFA) (0.1 mL, 1.3 mmol), and the obtained
mixture was stirred at room temperature overnight. Reaction mixture
was then concentrated under reduced pressure to dry and was
triturated with diethyl ether. The obtained yellow precipitate was then
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were combined, dried over Na2SO4, filtered, and concentrated. An oily
residue was obtained and was purified by preparative TLC on silica gel
(5% of methanol in dichloromethane) to afford compound 25 (128
mg, 45%). MS (ESI+): 593 [M + H]+.
2-(3-{[5-(2-Aminoethoxy)benzothiazol-2-ylmethyl]amino}-
[1,2,4]triazin-5-yl)-3-methylphenol Hydrochloride (26). To a
solution of 25 (128 mg, 0.21 mmol) in a mixture of dichloromethane
(1 mL) and methanol (0.2 mL) was added dropwise a 4 M solution of
HCl in dioxane (525 μL, 2.1 mmol) at 0 °C. The mixture was stirred
for 4 h at room temperature. The mixture was evaporated to dryness
to afford 26 as a white solid hydrochloride salt (53 mg, 62%). MS
(ESI+): 409 [M + H]+.
4-Ethyl-2,3-dioxopiperazine-1-carboxylic Acid [2-(2-{[5-(2-
Hydroxy-6-methylphenyl)[1,2,4]triazin-3-ylamino]methyl}-
benzothiazol-5-yloxy)ethyl]amide (2). To the solution of 26 (30
mg, 0.073 mmol) in the mixture of THF (0.7 mL) and DMF (0.4 mL)
were added DIPEA (39 μL, 0.22 mmol) and suspension of 4-ethyl-2,3-
dioxopiperazine-1-carbonyl chloride (34 mg, 0.16 mmol) in 0.5 mL of
THF at 0 °C under an argon atmosphere. The obtained mixture was
then stirred at room temperature overnight. The mixture was
evaporated to dryness, and the residue was partitioned between ice−
water and ethyl acetate. The organic layer was dried over Na2SO4,
filtered, and concentrated. The obtained oily residue was purified by
preparative TLC on silica gel (8% of methanol in dichloromethane) to
afford 2 as a yellow powder (12 mg, 29%). MS (ESI+): 577 [M + H]+.
1H MNR (400 MHz, MeOD) δ: 8.74 (1H, s); 7.82 (1H, d, J = 8.8
Hz); 7.51 (1H, d, J = 2.3 Hz); 7.21 (1H, t, J = 7.8 Hz); 7.13 (1H, dd,
J1 = 8.8 Hz, J2 = 2.3 Hz); 6.80 (2H, m); 5.10 (2H, s); 4.25 (2H, t, J =
5.3 Hz); 4.11 (2H, m); 3.82 (2H, t, J = 5.3 Hz); 3.67 (2H, m), 3.57
(2H, q, J = 7.2 Hz); 2.06 (3H, br s); 1.25 (3H, t, J = 7.2 Hz).
N-[2-(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]-
methyl}benzothiazol-5-yloxy)ethyl]-3,4-dimethoxybenzene-
sulfonamide (28). To a solution of 2710 (hydrochloride salt, 61 mg,
0.128 mmol) and DIPEA (91 μL, 0.512 mmol) in a mixture of dioxane
(1 mL) and DMF (1 mL) was added dropwise at 0 °C with stirring
under argon freshly prepared solution of 3,4-dimethoxybenzenesul-
fonyl chloride in DMF (1 mL). Then the reaction mixture was allowed
to heat to room temperature and was stirred at this temperature for 1
h. The reaction mixture was poured into ice (∼5 mL), extracted with
ethyl acetate, and washed with saturated aqueous solution of NH4Cl.
The combined organic phases were dried over Na2SO4 and
concentrated. The residue was purified by preparative TLC on silica
gel (5% of methanol in dichloromethane) to afford compound 28 as
white powder (64 mg, 78%). MS (ESI+): 645, 647 [M + H]+.
N-[2-(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]-
methyl}benzothiazol-5-yloxy)ethyl]-3,4-dihydroxybenzenesul-
fonamide (3). To a solution of 28 (26 mg, 0.045 mmol) in
dichloromethane (1 mL) under argon atmosphere at −78 °C was
added dropwise a 1 M solution of BBr3 (1.12 mmol) in dichloro-
methane, and the mixture was allowed to warm from −78 to −30 °C
over 2 h. Then the mixture was cooled to −50 °C, quenched by
methanol (1 mL), and allowed to warm to room temperature
overnight. The mixture was concentrated under reduced pressure,
partitioned between water and ethyl acetate, and then additionally
extracted with ethyl acetate. The combined organic layers were dried
over Na2SO4 and concentrated. The residue was purified by
preparative TLC on silica gel (6% of methanol in dichloromethane)
to afford compound 3 as beige powder (5.8 mg, 20%). MS (ESI+):
619, 621 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ: 8.75 (1H, s);
7.81 (1H, d, J = 8.8 Hz); 7.55 (3H, m); 7.41 (1H, br s); 7.33 (1H, d, J
= 2.2 Hz); 7.30 (1H, dd, J1 = 8.3 Hz, J2 = 2.2 Hz); 7.05 (1H, dd, J1 =
8.8 Hz, J2 = 2.4 Hz); 6.90 (1H, d, J = 8.3 Hz); 5.10 (2H, br s); 4.10
(2H, t, J = 5.5 Hz); 3.34 (2H, t, J = 5.5 Hz).
(20 mg, 0.128 mmol), and 4-dimethylaminopyridine (2 mg, cat.)
under argon atmosphere. The mixture was sonicated and then was
stirred at room temperature overnight, then concentrated under
reduced pressure, partitioned between water and ethyl acetate, and
then additionally extracted with ethyl acetate. The combined organic
layers were dried over Na2SO4 and concentrated. The residue was
purified by preparative TLC on silica gel (5% of methanol in
dichloromethane) to afford compound 8 as an off-white powder (25
1
mg, 30%). MS (ESI+): 652, 654 [M + H]+. H NMR (400 MHz,
DMSO-d6) δ: 12.11 (1H, br s); 9.20 (1H, br s); 8.87 (1H, s); 8.57
(1H, br s); 7.89 (1H, d, J = 8.5 Hz); 7.75−7.38 (6H, m); 7.05 (1H, d,
J = 8.5 Hz); 6.98 (2H, m); 5.35 (1H, t, J = 8.9 Hz); 5.04 (2H, br s);
4.14 (2H, t, J = 5.2 Hz); 3.69−3.55 (4H, m).
5-Hydroxy-4-oxo-4H-pyran-2-carboxylic Acid [2-(2-{[5-(2,6-
Dichlorophenyl)[1,2,4]triazin-3-ylamino]methyl}benzothiazol-
5-yloxy)ethyl]amide (5). MS (ESI+): 585, 587 [M + H]+. 1H NMR
(400 MHz, DMSO-d6) δ: 9.56 (1H, br s); 9.11 (1H, t, J = 5.3 Hz);
8.86 (1H, s); 8.12 (1H, s); 7.90 (1H, d, J = 8.6 Hz); 7.72−7.46 (5H,
m); 7.05 (1H, d, J = 7.0 Hz); 6.90 (1H, s); 5.20−4.80 (2H, m); 4.19
(2H, t, J = 5.1 Hz); 3.65 (2H, q, J = 5.4 Hz).
Methanesulfonic Acid 2-(2-{[5-(2,6-Dichlorophenyl)[1,2,4]-
triazin-3-ylamino]methyl}benzothiazol-5-yloxy)ethyl Ester
(30). To a solution of 2-(2-{[5-(2,6-dichlorophenyl)[1,2,4]triazin-3-
ylamino]methyl}benzothiazol-5-yloxy)ethanol 2910 (80 mg, 0.18
mmol) and triethylamine (28 μL, 0.2 mmol) in anhydrous THF (2
mL) at 0 C° under argon atmosphere was added dropwise mesyl
chloride (16 μL, 0.2 mmol). The mixture was stirred at 0 °C for 30
min and thereafter allowed to warm to room temperature over 2 h.
After concentration in vacuo and treatment with water and ethyl
acetate, the combined organic layers were washed with 0.05 M HCl,
NaHCO3, brine, dried over Na2SO4, and filtered. After concentration,
the yellowish oil product 30 (85 mg, 90% crude yield) was used as
such without further purification. MS (ESI+): 526, 528 [M + H]+.
3-[2-(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]-
methyl}benzothiazol-5-yloxy)ethylsulfanyl]-6-hydroxy-2-
methyl-2H-[1,2,4]triazin-5-one (7). A mixture of 1,2,5,6-tetrahydro-
5,6-dioxo-3-mercapto-2-methyl-as-triazine (44 mg, 0.28 mmol) and
triethylamine (20 μL, 0.14 mmol) in DMF (2 mL) was added to
freshly prepared 30 (47 mg, 0.09 mmol), and the obtained solution
was stirred at 60 °C for 72 h. Then the mixture was treated with ice−
water and ethyl acetate and extracted additionally with ethyl acetate,
dichloromethane, and butanol. The combined organic layers were
dried over Na2SO4 and filtered. The filtrate was concentrated, and the
residue was purified by preparative TLC on silica gel (6% of methanol
in dichloromethane) to afford compound 7 as a beige powder (15 mg,
1
28%). MS (ESI+): 589, 591 [M + H]+. H NMR (400 MHz, DMSO-
d6) δ: 13.18 (1H, br s); 9.15 (1H, br s); 8.86 (1H, s); 7.91 (1H, d, J =
8.4 Hz); 7.75−7.51 (4H, m); 7.09 (1H, d, J = 8.4 Hz); 5.04 (2H, br s);
4.45 (2H, t, J = 5.3 Hz); 4.41 (2H, t, J = 5.3 Hz), 3.72 (3H, s).
(3-{(3-tert-Butoxycarbonylaminopropyl)[2-(2-{[5-(2,6-
dichlorophenyl)[1,2,4]triazin-3-ylamino]methyl}benzothiazol-
5-yloxy)ethyl]amino}propyl)carbamic Acid tert-Butyl Ester
(31). A mixtute of [3-(3-tert-butoxycarbonylaminopropylamino)-
propyl]carbamic acid tert-butyl ester 3228 (58 mg, 0.18 mmol) and
30 (47 mg, 0.09 mmol) in acetonitrile (0.5 mL) was stirred at 85 °C
for 48 h. The mixture was concentrated in vacuo and treated with
water and ethyl acetate. The separated organic layer was washed with a
saturated aqueous solution of NH4Cl, dried over Na2SO4, and filtered.
The filtrate was concentrated and the residue was purified by
preparative TLC on silica gel (33% aqueous NH3/methanol/
dichloromethane 0.7/7/93) to afford compound 31 as a colorless oil
(30 mg, 43%). MS (ESI+): 761, 763 [M + H]+.
N1-(3-Aminopropyl)-N1-[2-(2-{[5-(2,6-dichlorophenyl)[1,2,4]-
triazin-3-ylamino]methyl}benzothiazol-5-yloxy)ethyl]-
propane-1,3-diamine (17). To a solution of 31 (30 mg, 0.39 mmol)
in dichloromethane (0.7 mL) was added 4 N solution of HCl in
dioxane (0.3 mL, 1.2 mmol) at 0 °C under argon atmosphere, and the
mixture was stirred at room temperature for 3 h. The obtained
hydrochloride was desalted by purification through Agilent SCX
cartridge, using DCM, then MeOH, then 1 M ammonia solution in
MeOH as eluents, affording 17 as a colorless oil (5.1 mg, 23%). MS
(R)-2-(2-Hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic
Acid [2-(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]-
methyl}benzothiazol-5-yloxy)ethyl]amide (8). To a thin suspen-
sion of 2710 (hydrochloride salt, 57 mg, 0.127 mmol) and DIPEA (44
μL, 0.25 mmol) in dichloromethane (1 mL) were added (R)-2-(2-
hydroxyphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid45 (33 mg,
0.15 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDAC, 48 mg, 0.25 mmol), 1-hydroxybenzotriazol hydrate
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Journal of Medicinal Chemistry
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1
(ESI+): 561, 563 [M + H]+. H NMR (400 MHz, MeOD) δ: 8.74
with methanol (1 mL). The solution was diluted with saturated
NaHCO3 aqueous solution (5 mL) and extracted with ethyl acetate (3
× 2 mL). The combined organic layers were washed with brine, dried
over Na2SO4, and concentrated in vacuum and the crude was purified
by preparative TLC on silica gel (15% of methanol in dichloro-
methane) to give compound 10 (4.6 mg, 44%) as a yellow solid. MS
(ESI+): 450 [M + H]+. 1H MNR (400 MHz, MeOD) δ: 8.52 (1H, s);
7.68(1H, d, J =8.7 Hz); 7.29 (1H, d, J = 2.1 Hz); 6.92 (1H, dd,, J1 =
8.7 Hz, J2 = 2.4 Hz); 6.38 (1H, s); 5.00 (2H, br s); 2.79 (2H, t, J = 6.3
Hz); 2.48 (6H, s); 2.00−1.90 (6H, m).
(1H, s); 7.83 (1H, d, J = 8.8 Hz); 7.59−7.48 (4H, m); 7.12 (1H, dd, J1
= 8.8 Hz, J2 = 2.4 Hz); 5.11 (2H, br s); 4.23 (2H, t, J = 5.5 Hz); 2.99
(2H, t, J = 5.5 Hz); 2.75 (4H, t, J = 7.0 Hz); 2.69 (4H, t, J = 7.1 Hz);
1.74 (4H, quint, J = 7.1 Hz).
[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-yl]-{5-[2-(4-methylpi-
perazin-1-yl)ethoxy]benzothiazol-2-ylmethyl}amine (13). MS
1
(ESI+): 530, 532 [M + H]+. H NMR (400 MHz, CDCl3) δ: 8.70
(1H, s); 7.68 (1H, d, J = 8.8 Hz); 7.45 (2H, dd, J1 = 11.8 Hz, J2 = 10.3
Hz); 7.42 (1H, s); 7.35 (1H, dd, J1 = 9.1 Hz, J2 = 6.8 Hz); 7.04 (1H,
dd, J1 = 8.8 Hz, J2 = 6.4 Hz); 6.50−6.25 (1H, br s); 5.22−5.12 (2H, br
s); 4.18 (2H, t, J = 5.8 Hz); 2.86 (2H, t, J = 5.7 Hz); 2.72−2.56 (4H,
br s); 2.56−2.40 (4H, br s); 2.30 (3H, s).
2-(2-{[5-(2,6-Dichlorophenyl)[1,2,4]triazin-3-ylamino]-
methyl}benzothiazol-5-yloxy)-N-prop-2-ynylacetamide (34).
To acid 1910 (100 mg, 0.22 mmol) were added 1-hydroxybenzotriazol
(34 mg, 0.22 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide hydrochloride (85 mg, 0.44 mmol), and dry dichloromethane (1
mL). The mixture was stirred for 1 h at room temperature. Then to
the mixture were added diisopropylethylamine (78 μL, 0.44 mmol)
and propargylamine (24 mg, 0.44 mmol). The mixture was stirred
overnight at room temperature and then partitioned between water
and ethyl acetate. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by preparative TLC on silica
gel (5% of methanol in dichloromethane) to afford 34 (48 mg, 44%)
as a white powder. MS (ESI+): 499, 501 [M + H]+.
N , N ′ - [ ( 1 R , 3 S , 4 R , 5 R , 6 S ) - 4 - ( { 6 - D e o x y - 6 - [ ( 2 , 2 -
dimethylpropanoyl)amino]-α-D-glucopyranosyl}oxy)-6-({3,6-
dideoxy-6-(4-{[({[2-({[5-(2,6-dichlorophenyl)-1,2,4-triazin-3-yl]-
amino}methyl)-1,3-benzothiazol-5-yl]oxy}acetyl)amino]-
methyl}-1H-1,2,3-triazol-1-yl)-3-[(2,2-dimethylpropanoyl)-
amino]-α-D-glucopyranosyl}oxy)-5-hydroxycyclohexane-1,3-
diyl]bis(2,2-dimethylpropanamide) (35). In an amber flask N-
protected 6″-azido-kanamycin A 3329 (80 mg, 0.088 mmol) and
alkyne 34 (44 mg, 0.088 mmol) were diluted in tert-butanol (0.5 mL).
To this solution were added a solution of copper sulfate (0.14 mg,
0.0009 mmol) and sodium ascorbate (2 mg, 0.009 mmol) in water
(0.5 mL). Then the mixture was stirred for 3 days at room
temperature. The mixture was partitioned between water and ethyl
acetate. The aqueous layer was extracted with ethyl acetate. Then the
combined organic layers were dried over Na2SO4 and concentrated in
vacuum. The crude was purified by chromatography on silica gel
(gradient 3−15% of methanol in dichloromethane) to afford
compound 35 (15.4 mg, 12%) as a yellow solid. MS (ESI+): 1408−
1410 [M + H]+. 1H MNR (400 MHz, MeOD) δ: 8.69 (1H, s); 7.85−
7.83 (2H, m); 7.50 (4H, br s); 7.19 (1H, dd,, J1 = 8.8 Hz, J2 = 6.4 Hz);
6.7−6.4 (3H, m); 5.17−4.95(4H, m); 4.75−4.35 (7H, m); 3.75−3.00
(18H, m); 3.00 (1H, br s); 1.48−1.38 (36H, m).
2-Dimethylamino-N-(4-{3-[(5-hydroxybenzothiazol-2-
ylmethyl)amino][1,2,4]triazin-5-yl}-3,5-dimethylphenyl)-
1
acetamide (11). MS (ESI+): 464 [M + H]+. H NMR (400 MHz,
DMSO-d6) δ ppm: 9.70 (1H, br s); 9.64 (1H, s); 9.00−8.50 (2H, m);
7.76 (1H, d, J = 8.7 Hz); 7.43 (2H, br s); 7.25 (1H, s); 6.88 (1H, dd, J1
= 8.9 Hz, J2 = 6.6 Hz); 4.90 (2H, br s); 3.06 (2H, s); 2.27 (6H, s);
2.10−1.80 (6H; m).
N-[4-(3-{[5-(2-Aminoethoxy)benzothiazol-2-ylmethyl]-
amino}[1,2,4]triazin-5-yl)-3,5-dimethylphenyl]-N′,N′-dimethyl-
ethane-1,2-diamine (12). (a) A solution of [4-(3-chloro[1,2,4]-
triazin-5-yl)-3,5-dimethylphenyl]carbamic acid tert-butyl ester 38 (160
mg, 0.48 mmol),10 tert-butyl 2-{[2-(aminomethyl)-1,3-benzothiazol-5-
yl]oxy}ethylcarbamate 24 (180 mg, 0.56 mmol),10 and diisopropyle-
thylamine (84 μL, 0.48 mmol) in anhydrous acetonitrile (2 mL) was
stirred under argon at 85 °C overnight. An aqueous solution of
ammonium chloride was added, and the reaction mixture was extracted
with ethyl acetate. The combined organic extracts were dried over
sodium sulfate, filtered, and evaporated. Purification by preparative
TLC (silica gel, dichloromethane/methanol 95/5) afforded tert-butyl
[4-(3-{[5-(2-tert-butoxycarbonylaminoethoxy)benzothiazol-2-
ylmethyl]amino}[1,2,4]triazin-5-yl)-3,5-dimethylphenyl]carbamate
(174.1 mg, 58%) as an orange solid. ESI-MS m/z 622 (M + H)+.
(b) (i) A mixture of 37% aqueous hydrochloric acid (1.25 mL) and
ethyl acetate (3.75 mL) was added to tert-butyl [4-(3-{[5-(2-tert-
butoxycarbonylaminoethoxy)benzothiazol-2-ylmethyl]amino}[1,2,4]-
triazin-5-yl)-3,5-dimethylphenyl]carbamate (174.1 mg, 0.28 mmol),
and the mixture was cooled to 0 °C. The reaction mixture was stirred
for 1 h allowing the temperature to rise to room temperature. A dilute
aqueous solution of potassium hydroxyde was added until basic pH
was reached. The solution was extracted with ethyl acetate and
dichloromethane, and the combined organic extracts were dried over
sodium sulfate, filtered, and evaporated. (ii) The crude product was
dissolved in anhydrous THF (3 mL), and triethylamine (60 μL, 0.43
mmol) and di-tert-butyl dicarbonate (67.2 mg, 0.31 mmol) were
added. The resulting mixture was stirred at room temperature
overnight. An aqueous solution of ammonium chloride was added,
and the reaction mixture was extracted with diethyl ether and ethyl
acetate. The combined organic extracts were dried over sodium sulfate,
filtered, and evaporated. Purification by preparative TLC (silica gel,
dichloromethane/methanol 93/7) afforded tert-butyl [2-(2-{[5-(4-
amino-2,6-dimethylphenyl)[1,2,4]triazin-3-ylamino]methyl}-
benzothiazol-5-yloxy)ethyl]carbamate (107 mg, 73%) as a yellow solid.
ESI-MS m/z 522 (M + H)+.
(c) (i) Under argon, 2-chloro-N,N-dimethylethanamine hydro-
chloride (59 mg, 0.41 mmol), sodium hydrogencarbonate (34.5 mg,
0.41 mmol), and a catalytic amount of sodium iodide were successively
added to a solution of tert-butyl [2-(2-{[5-(4-amino-2,6-
dimethylphenyl)[1,2,4]triazin-3-ylamino]methyl}benzothiazol-5-
yloxy)ethyl]carbamate (107 mg, 0.21 mmol) in ethanol (1.65 mL).
The reaction mixture was stirred at room temperature for 1 h and then
at 80 °C overnight. The solution was then dried over potassium
carbonate, filtered, and concentrated. Purification by preparative TLC
(silica gel, dichloromethane/methanol 85/15) led to {2-[2-({5-[4-(2-
dimethylaminoethylamino)-2,6-dimethylphenyl][1,2,4]triazin-3-
ylamino}methyl)benzothiazol-5-yloxy]ethyl}carbamic acid tert-butyl
ester. (ii) The latter compound was dissolved in methanol (125
μL), and a solution of hydrogen chloride in dioxane (4 N, 375 μL, 1.5
mmol) was added. The resulting mixture was stirred at room
temperature for 25 min. Methanol and dichloromethane were added,
and the solution was dried over potassium carbonate, filtered, and
(1S,2R,3R,4S,6R)-4,6-Diamino-3-[(6-amino-3-[(6-amino-6-
deoxy-α-D-glucopyranosyl)oxy]-2-hydroxycyclohexyl-3-
amino-3,6-dideoxy-6-(4-{[({[2-({[5-(2,6-dichlorophenyl)-1,2,4-
triazin-3-yl]amino}methyl)-1,3-benzothiazol-5-yl]oxy}acetyl)-
amino]methyl}-1H-1,2,3-triazol-1-yl)-α-D-glucopyranoside (9).
Compound 35 (15 mg, 0.010 mmol) was dissolved in methanol
(0.5 mL), and HCl 4 M in dioxane (106 μL, 0.424 mmol) was added.
The mixture was stirred overnight at room temperature and then
concentrated in vacuo. The crude was dissolved in methanol (0.4 mL)
and precipitated with diethyl ether (2 mL). The solid was isolated by
filtration, washed with ether, and dried in vacuum. Compound 9 (7.8
mg, 64%) was obtained as a light yellow solid. MS (ESI+): 1008−1010
[M + H]+. H MNR (400 MHz, MeOD) δ: 8.70 (1H, s); 7.90−7.75
(2H, m); 7.60−7.44 (4H, m); 7.21 (1H, d, J = 8.2 Hz); 5.56−5.46
(2H, m); 5.16−5.06 (3H, m); 4.64−3.36 (20H, m); 3.14−3.02 (2H,
m); 2.47 (1H, s); 1.97 (1H, s).
2-({5-[4-(2-Dimethylaminoethylamino)-2,6-dimethylphenyl]-
[1,2,4]triazin-3-ylamino}methyl)benzothiazol-5-ol (10). Com-
pound 3610 (10.9 mg, 0.023 mmol) was dissolved in dichloromethane
(0.5 mL) and cooled to −78 °C. Boron tribromide 1 M in
dichloromethane (253 μL, 0.253 mmol) was added dropwise, and
the mixture was stirred for 1 h at this temperature and then for 4 h at
room temperature. The mixture was cooled to −78 °C and quenched
1
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Journal of Medicinal Chemistry
Article
concentrated. The crude product was purified by preparative TLC
(silica gel, dichloromethane/methanol/aqueous ammonium hydroxide
33%, 90/10/2) to afford compound 12 (13.6 mg, 13%) as a yellow
(15) Vaara, M. Agents that increase the permeability of the outer
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Miller, M. H. Tobramycin uptake in Escherichia coli membrane vesicles.
Antimicrob. Agents Chemother. 1995, 39, 467−475.
1
solid. ESI-MS m/z 493 (M + H)+. H NMR (DMSO-d6), δ (ppm):
8.63 (s, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.07
(dd, J = 8.8 and 2.0 Hz, 1H), 6.32 (br s, 2H), 4.93 (br s, 2H), 4.16−
4.13 (m, 2H), 3.12−3.10 (m, 4H), 2.44−2.40 (m, 2H), 2.18 (s, 6H),
1.91 (br s, 6H).
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accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and
Escherichia coli. J. Antimicrob. Chemother. 1999, 43, 61−70.
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OmpF channel facilitates their translocation. Biophys. J. 2006, 90,
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(19) Mach, T.; Neves, P.; Spiga, E.; Weingart, H.; Winterhalter, M.;
Ruggerone, P.; Ceccarelli, M.; Gameiro, P. Facilitated permeation of
antibiotics across membrane channelsinteraction of the quinolone
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AUTHOR INFORMATION
Corresponding Author
*Phone: +33-157140522. Fax: +33-157140524. E-mail: vincent.
gerusz@mutabilis.fr.
■
Notes
The authors declare no competing financial interest.
(20) Nikaido, H.; Thanassi, D. G. Penetration of lipophilic agents
with multiple protonation sites into bacterial cells: tetracyclines and
fluoroquinolones as examples. Antimicrob. Agents Chemother. 1993, 37,
1393−1399.
ACKNOWLEDGMENTS
■
We thank Prof. Murray Junop and his team for the delivery of
co-structures of a few nonvectorized inhibitors in HldA. We are
also grateful to Zyfine for the synthesis of building blocks and
to Dr. Franco̧ is Bellamy, Prof. Xavier Nassif, and Dr. Patrice
Renaut for stimulating discussions on these topics.
́ ́ ́ ́
(21) Takacs-Novak, K.; Jozan, M.; Szasz, G. Lipophilicity of
amphoteric molecules expressed by the true partition coefficient. Int.
J. Pharm. 1995, 113, 47−55.
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Annu. Rev. Microbiol. 1984, 38, 237−264.
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