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. 2013 Dec 4;5(1):94–97. doi: 10.1021/ml400457j

(S)-N-Methyldihydroquinazolinones are the Active Enantiomers of Retro-2 Derived Compounds against Toxins

Neetu Gupta †, Valérie Pons ‡, Romain Noël ‡, David-Alexandre Buisson ‡, Aurélien Michau †, Ludger Johannes §,⊥, Daniel Gillet †,*, Julien Barbier †, Jean-Christophe Cintrat ‡,*
PMCID: PMC4027625  PMID: 24900779

Abstract

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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

Ricin is a highly toxic protein abundant in castor beans and classified as a potential bioweapon. After ingestion or 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

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 dihydroquinazolinones was bioactive, but its absolute configuration was unsolved.

Scheme 1. Previous Studies on Retro-2 Optimization and Objectives of This Work (with EC50 against Shiga Toxin).

Scheme 1

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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.

During the course of our previous SAR study starting from Retro-2, we obtained N-methylated cyclic analogues leading to a potent inhibitor of Stx action in cells (compound 94, EC50 = 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.

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.

Scheme 2

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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 1.

Figure 1

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Chiral HPLC chromatograms of rac-Retro-2.1 and purified enantiomers, Retro-2.1a and Retro-2.1b.

To unequivocally assign the absolute configuration of each enantiomer, crystal structure determinations were obtained on separate enantiomers. Among the different crystallization 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.

Figure 2.

Figure 2

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Structure determination of the Retro-2.1 enantiomers by X-ray crystallography.

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).

Figure 3.

Figure 3

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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.

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 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 pharmacological 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 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

Glossary

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 network

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

Supporting Information Available

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 Contributions

∥ N.G. and V.P. contributed equally to this work.

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.

The authors declare no competing financial interest.

Supplementary Material

ml400457j_si_001.pdf (289.3KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml400457j_si_001.pdf (289.3KB, pdf)

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