(S)-N-methyldihydroquinazolinones are the active enantiomers of retro-2 derived compounds against toxins

Article abstract of DOI:10.1021/ml400457j

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of a new compound, named Retro-2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM, respectively.

Full text of DOI:10.1021/ml400457j

Letter
pubs.acs.org/acsmedchemlett
(S)‑N‑Methyldihydroquinazolinones are the Active Enantiomers of
Retro‑2 Derived Compounds against Toxins
Neetu Gupta,†,∥ Valerie Pons,‡,∥ Romain Noel,‡ David-Alexandre Buisson,‡ Aurelien Michau,†
́
́
̈
Ludger Johannes,§,⊥ Daniel Gillet,†,* Julien Barbier,† and Jean-Christophe Cintrat‡,*
†CEA, iBiTec-S/SIMOPRO, CEA-Saclay, LabEx LERMIT, F-91191 Gif-sur-Yvette, France
‡CEA, iBiTec-S/SCBM, CEA-Saclay, LabEx LERMIT, F-91191 Gif-sur-Yvette, France
§U1143 INSERM, 75005 Paris, France
⊥Institut Curie, Centre de Recherche, Chemical Biology of Membranes and Therapeutic Delivery, 26 Rue d’Ulm, 75248 Paris Cedex
05, France
S
* Supporting Information
ABSTRACT: This study reports the synthesis, chromatographic separation, and
pharmacological evaluation of the two enantiomers of a new compound, named Retro-
2.1, active against toxins by inhibiting intracellular trafficking via the retrograde route. The
absolute configuration of the bioactive enantiomer has been assigned from X-ray diffraction
to the (S)-enantiomer. To date, (S)-Retro-2.1 is the most potent molecule to counteract
the cytotoxic potential of ricin and Shiga toxin, with EC50 values of 23 and 54 nM,
respectively.
KEYWORDS: Retro-2.1, Shiga toxin, ricin, dihydroquinazolinones
icin is a highly toxic protein abundant in castor beans and
classified as a potential bioweapon. After ingestion or
We have recently identified a compound named Retro-2 that
protects human cells against ricin and Stx by blocking
retrograde toxin transport at the early endosomes-TGN
interface.12 Retro-2 also protects mice against lethal ricin
challenge.12 A structure−activity relationship (SAR) study of
Retro-2 led to an improved activity against Stx up to 100-fold
for compounds sharing a dihydroquinazolinone scaffold
(cyclized Retro-2).13,14 We also demonstrated that only one
enantiomer (compound 62,13 Scheme 1) of dihydroquinazo-
linones was bioactive, but its absolute configuration was
unsolved.
Here, we report the synthesis and evaluation of a new
enantiopure dihydroquinazolinone compound, named Retro-
2.1, acting against Stx cytotoxicity with improved potency
(∼500-fold compared to Retro-2) and demonstrate that Retro-
2.1 is also effective against ricin in vitro (∼1000-fold increased
activity compared to Retro-2). We establish from crystal X-ray
diffraction data that the antitoxin activity resides mainly in the
S-enantiomer.
R
inhalation, ricin causes major organ damages that can ultimately
lead to the death of the exposed organism. To date no
approved treatments or prophylaxis for human use is available.
The Shiga toxin (Stx) family consists of related protein toxins
that are produced by the bacteria Shigella dysenteriae and certain
pathogenic strains of Escherichia coli.1,2 Bacteria producing
these toxins are responsible for a number of diseases such as the
life-threatening complication hemolytic uremic syndrome
(HUS).3 In 2011, a major outbreak caused by E. coli
O104:H4 infected about 4000 people in Europe, causing
more than 900 cases of HUS resulting in 54 deaths.4 So far, no
effective therapy exists for Stx intoxication.5,6 Ricin and Stx
share several characteristics. They have one moiety (B-subunit)
that binds to their respective cellular receptors (glycoproteins
and glycolipids for ricin; the glycosphingolipid Gb3 for Stx),
while another moiety (A-subunit) enters the cytosol and
inactivates protein biosynthesis. Both toxins are transported in
a retrograde manner from the plasma membrane via endosomes
and the TGN to the endoplasmic reticulum (ER),7 before
translocation to the cytosol, where they enzymatically inactivate
the 28S RNA of the 60S ribosomal subunit (reviewed in refs
7−10). Inhibitors acting on the intracellular routing of these
toxins likely offer novel options for the development of
therapeutic strategies.11
During the course of our previous SAR study starting from
Retro-2, we obtained N-methylated cyclic analogues leading to
Received: November 12, 2013
Accepted: December 4, 2013
Published: December 4, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Scheme 1. Previous Studies on Retro-2 Optimization and Objectives of This Work (with EC50 against Shiga Toxin)
Scheme 2. Synthesis of 6-Fluoro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-
one, Retro-2.1
a potent inhibitor of Stx action in cells (compound 94, EC50
=
conditions that we screened, a mixture of CH2Cl2 and methanol
(1:1) proved efficient to obtain monocrystals suitable for X-ray
diffraction (slow evaporation method). These studies revealed
that Retro-2.1a had a S-stereochemistry at the C-2 position of
the dihydroquinazolinone core (Figure 2), while Retro-2.1b was
the R-isomer.
0.3 μM,13 Scheme 1). In parallel, substitution of the phenyl ring
of the dihydroquinazolinone scaffold of 62 was evaluated.
Among all the substituents, only the 6-fluoro derivative showed
a better activity compared to the parent Retro-2cycl (EC50 = 11
μM vs 27 μM, respectively)13 (Scheme 1). We describe here
the synthesis and evaluation of a compound combining the best
pharmacophores.
The synthesis of compound 5 was achieved according to our
previously published procedure starting from the commercially
available 4-fluoro anthranilic acid 1 (Scheme 2) in 39% yield
over 4 steps.
In addition, since only one enantiomer was active on
previous dihydroquinazolinones synthesized as racemates,13
a
chiral phase HPLC separation of Retro-2.1 was performed on a
ChiralPak IB column (see Experimental Section and Figure 1).
No contamination of Retro-2.1a by Retro-2.1b or vice versa
could be identified on the chromatograms (Figure 1).
Figure 2. Structure determination of the Retro-2.1 enantiomers by X-
ray crystallography.
To unequivocally assign the absolute configuration of each
enantiomer, crystal structure determinations were obtained on
separate enantiomers. Among the different crystallization
The biological activity of Retro-2.1 and each enantiomer was
then evaluated against Stx and ricin (Figure 3). Retro-2.1
racemate inhibited Stx action on HeLa cells with an EC50 of
102
15.0 nM (Figure 3, upper left panel; n = 9) more
potently than the previously described best compound
(compound 94,13 EC50 = 0.3 μM). It also inhibited ricin
induced-toxicity on human pulmonary A549 cells with an EC50
of 57 2.4 nM (Figure 3, upper right panel; n = 3).
When tested separately, Retro-2.1a ((S)-Retro-2.1) potently
inhibited toxin cytotoxicity (eutomer), while the second eluted
compound, Retro-2.1b ((R)-Retro-2.1), only showed moderate
activity at micromolar concentrations (distomer). Indeed,
(S)-Retro-2.1 inhibited both Stx cellular action (EC50 of 54
16 nM; Figure 3, middle left panel; n = 7) and ricin (EC50 of 23
4 nM; Figure 3, middle right panel; n = 4), whereas
(R)-Retro-2.1 exhibited only an EC50 of 2.4 1 μM against Stx
(Figure 3, lower left panel; n = 4) and an EC50 of 3.2 0.1 μM
against ricin (Figure 3, lower right panel; n = 3). These results
Figure 1. Chiral HPLC chromatograms of rac-Retro-2.1 and purified
enantiomers, Retro-2.1a and Retro-2.1b.
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Figure 3. Retro-2.1 protects cells against Stx and ricin. Intoxication of HeLa cells by Stx (left) and intoxication of A549 cells by ricin (right) in the
presence of inhibitors. Cells were incubated 1 h at 37 °C with DMSO (0.04%, black data points in all parts of the figure), rac-Retro-2.1 (various
concentrations; upper panel), (S)-Retro-2.1 enantiomer (various concentrations; middle panel), or (R)-Retro-2.1 enantiomer (various
concentrations; lower panel) before addition of toxins at the indicated concentrations. Each data point represents the mean of duplicate SEM
of a representative experiment. EC50 values are the mean of n independent experiments (see text for more details). Note that the concentrations used
for racemate and enantiomers are different.
are in accordance with the EC50 values obtained from the
racemate form with a 2-fold increase of potency of the eutomer.
Eudismic ratios of 45 and 135, respectively for Stx and ricin,
were calculated from the EC50 values of cytotoxicity assays
reported in this paper, reflecting a high enantioselectivity. This
eudismic ratio may also explain the absence of activity of the
distomer of compound 62 reported previously13 on Stx, since
the highest concentration tested for the distomer was 30 μM
(due to low solubility) while the EC50 of the racemate was 10
μM.
It is worth noting that besides a lower potency, determined
by a higher EC50, (R)-Retro-2.1 also displayed a reduced
efficacy (lower maximal R-value) in comparison to (S)-Retro-
2.1 against both Stx and ricin (for Stx, R(S) = 357 vs R(R) = 68, n
= 6; for ricin, R(S) = 5 vs R(R) = 2.9, n = 3; R values are from
experiments performed in parallel). These differential pharma-
cological activities proved that only the (S)-enantiomer fully
retained the desired antitoxin activity.
Retro-2.1 has a stronger efficacy (R-values) against Stx than
against ricin, as observed for Retro-2.12 Most likely, ricin can
use several mechanisms to traffic into cells by binding to a
variety of glycoreceptors. In contrast, Stx only binds to Gb3 and
may be facing limited molecular redundancy for reaching TGN
membranes. Ricin has also been proposed to cross endosomal
membranes to reach the cytosol, as an alternative mechanism to
retrotranslocation from the endoplasmic reticulum.15 This type
of endosomal escape would allow bypassing a Retro-2.1-
induced retrograde transport block.
We describe herein the synthesis and chromatographic
separation of Retro-2.1 enantiomers. In vitro experiments
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(3) Tarr, P. I.; Gordon, C. A.; Chandler, W. L. Shiga-toxin-producing
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proved the configuration of the stereocenter in position 2 is
crucial for activity, since one enantiomer is active to protect
cells against Stx and ricin. The absolute stereochemistry of the
eutomer has been assigned by X-ray diffraction. The (S)-isomer
proved to be the bioactive enantiomer, and (S)-Retro-2.1 is the
most active compound against these toxins reported so far.
Since N-methyldihydroquinazolinones derived from Retro-2
block the retrograde pathway within cells, optimized (S)-Retro-
2.1 has the potential to be developed as a broad-spectrum
antidote to a wide array of pathogens, including toxins and
parasites, as demonstrated previously in vitro and in vivo for
Retro-2.12,16
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ASSOCIATED CONTENT
■
S
* Supporting Information
General procedures, characterization of new compounds,
crystal structure determination of compound 5, chemicals for
in vitro experiments, intoxication assays, and determination of
EC50 values. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*For D.G.: phone, + 33 1 69 08 76 46; fax, + 33 1 69 08 90 71;
e-mail, daniel.gillet@cea.fr.
*For J.-C.C.: phone, + 33 1 69 08 21 07; fax, + 33 1 69 08 79
91; e-mail, jean-christophe.cintrat@cea.fr.
Author Contributions
∥N.G. and V.P. contributed equally to this work.
Funding
This work (Project RetroScreen) has been funded by the
French National Agency for Research (ANR) under Contract
ANR-11-BSV2-0018, the Joint ministerial program of R&D
against CBRNe risks and CEA. N.G. was funded by the CEA
international PhD program.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
Stx, Shiga toxin; E. coli, Escherichia coli; HUS, hemolytic uremic
syndrome; Gb3, globotriaosylceramide; ER, endoplasmic
reticulum; RNA, ribonucleic acid; SAR, structure−activity
relationship; EC50, 50% effective concentration; HPLC, high
performance liquid chromatography; TGN, trans-Golgi net-
work; DMSO, dimethyl sulfoxide; SEM, standard error of the
mean; RP-HPLC, reversed phase−high performance liquid
chromatography; THF, tetrahydrofuran; DMF, dimethyl
formamide; PTSA, para-toluene sulfonic acid; NMR, nuclear
magnetic resonance; MS, mass spectrometry; HRMS, high
resolution mass spectrometry; LC/MS, liquid chromatography
coupled to mass spectrometry; IR, infrared; UV, ultraviolet;
DAD, diode array detector; DMEM, Dulbecco’s modified
Eagle’s medium; IC50, 50% inhibition concentration; TLC,
thin-layer chromatography
(15) Beaumelle, B.; Alami, M.; Hopkins, C. R. ATP-dependent
translocation of ricin across the membrane of purified endosomes. J.
Biol. Chem. 1993, 268, 23661−23669.
(16) Canton, J.; Kima, P. E. Targeting host syntaxin-5 preferentially
blocks Leishmania parasitophorous vacuole development in infected
cells and limits experimental Leishmania infections. Am. J. Pathol.
2012, 181, 1348−1355.
REFERENCES
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