NEW DIHYDROQUINAZOLINONES EXHIBITING PROTECTIVE ACTIVITY AGAINST INTRACELLULAR-ACTING TOXINS, INTRACELLULAR VIRUSES AND BACTERIA

Abstract

The invention relates to a new family of compounds of the type 2,3-dihydroquinazolin-4(1H)-one and the use thereof as inhibitors of the toxic effects of intracellular-acting toxins, such as ricin, Shiga toxins or the cholera toxin, using retrograde transport to intoxicate the cells, or viruses or bacteria using retrograde and/or syntaxin 5-dependent transport to infect the cells, specifically viruses or bacteria entering into the cells by means of endocytosis, or intracellular parasites.

Claims

1. A compound of general formula (I): ##STR00031## where: p is equal to 1, 2 or 3; R.sub.1 represents on each occurrence, independently, a hydrogen atom, a halogen atom, an alkoxy radical of 1 to 3 carbon atoms, in particular a methoxy group, —NO.sub.2 or —NH.sub.2; R.sub.2 and R.sub.3 independently of each other represent a group selected from H, —OH, —OR.sub.4, —NH.sub.2, —NHR.sub.5, —SO.sub.2—NH.sub.2, —SO.sub.2—NH—R.sub.6, on the condition that at least one of R.sub.2 and R.sub.3 does not represent H; R.sub.4, R.sub.5 and R.sub.6 independently of each other represent a group of formula (II) -L-(X).sub.i—(PEG)-(Y).sub.j—Z, wherein: i and j independently of each other represent 0 or 1; L represent —C(═O)— or —C(═O)—(CH.sub.2).sub.k—C(═O)—, where k is 1, 2 or 3, in particular 2; X and Y independently of each other represent a poly(lactic acid) or a poly(lactic acid-co-glycolic acid); PEG represents a poly(ethylene glycol); Z represents a group selected from H, a C.sub.1 to C.sub.3 alkyl, —OH, a O—C.sub.1 to C.sub.3 alkyl, or -L-R.sub.e, wherein L is defined as above and R.sub.e is a residue of formula (I) linked to said -L-(X).sub.i—(PEG)-(Y).sub.j— group defined above via its R.sub.2 or R.sub.3 group, said R.sub.2 or R.sub.3 group being —OH, —NH.sub.2 or —SO.sub.2—NH.sub.2; as well as the stereoisomeric forms, the mixtures of stereoisomeric forms or their pharmaceutically acceptable salts.

2. The compound of general formula (I) according to claim 1, wherein R.sub.3 represents a hydrogen atom.

3. The compound of general formula (I) according to any one of the preceding claims, wherein R.sub.2 represents a hydrogen atom.

4. The compound of general formula (I) according to any one of the preceding claims, wherein p is equal to 1.

5. The compound of general formula (I) according to any one of the preceding claims, wherein R.sub.1 represents a halogen atom, in particular a fluorine atom.

6. The compound of general formula (I) according to claim 1, of the following formula (Ia): ##STR00032##

7. The compound of general formula (I) according to claim 1, selected from: ##STR00033## ##STR00034##

8. The compound of general formula (I) as defined in any one of claims 1 to 7, for use in the prevention and/or treatment of disorders induced by the intracellular-acting toxins using the retrograde transport, or by the viruses or bacteria using the retrograde and/or syntaxin 5-dependent transport to infect the cells, in particular the viruses or bacteria entering the cells by endocytosis, or by the intracellular parasites.

9. The compound for use according to claim 8, characterised in that said intracellular-acting toxins are selected from the ricin, the Shiga toxin and the Shiga-like toxins (Stxs) produced by Shigella dysenteriae (Stx) and E. coli (Stx1 and Stx2), the cholera toxin (Ctx from Vibrio cholerae responsible for cholera), the pertussis toxin (Bordetella pertussis agent of whooping cough), the subtilase cytotoxin and the heat-labile enterotoxin (E. coli).

10. The compound for use according to claim 8, characterised in that said viruses are the pox viruses, in particular the smallpox virus, the monkeypox virus, the Vaccinia virus and the leporipoxviruses, in particular the myxomatosis virus, the cytomegaloviruses, the adeno-associated viruses, in particular of serotype 2, the polyomaviruses, in particular the polyomavirus JC and the polyomavirus BK, the papillomaviruses, the filoviruses, in particular the Ebola viruses and the Marburg virus, the enteroviruses, in particular the enterovirus 71, the herpes viruses, in particular the Herpes simplex virus of type 2, the viruses of the genus Arenavirus, in particular the lymphocytic choriomeningitis virus, the influenza viruses, in particular the influenzaviruses A, the pneumoviruses, in particular the respiratory syncytial virus.

11. A pharmaceutical composition or medicament comprising at least one compound of general formula (I) as defined in any one of claims 1 to 7 as active principle, and a pharmaceutically acceptable carrier, said pharmaceutical composition or said medicament being adapted in particular for an administration by the aerial, oral, parenteral or local routes.

Description

DESCRIPTION OF FIGURES

[0089] FIG. 1 shows the crystallographic structures of the ricin (A, whose name in the crystallographic structure database is pdb 2AAI) and the Stx2 (B, pdb 1R4P).

[0090] FIG. 2 is a schematic representation of the cell assay implemented in the experimental part.

[0091] FIG. 3 illustrates the IC.sub.50 calculation method performed in part II of the examples; the toxicity curves are performed in the absence of inhibitor (Control) and then in the presence of inhibitor at different concentrations.

EXAMPLES

I. Synthesis of Compounds According to the Invention

[0092] All chemicals and solvents used in the syntheses are of reactive grade and were used without additional purification. The CH.sub.2Cl.sub.2 was distilled over calcium hydride before use. The glassware was flame dried under vacuum and cooled under nitrogen to room temperature. All the reactions were carried out under dry nitrogen and monitored by TLC.

[0093] In particular, the purification was performed on a CombiFlash with a UV-vis detector and RediSep columns. The samples were adsorbed onto the Celite or the silica and loaded into solid-filled cartridges. In particular, an ethyl acetate/cyclohexane or methanol/dichloromethane gradient was used. The fractions were in particular collected on the basis of a detection at 254 nm.

[0094] The analysis and the purification by HPLC-MS was carried out using a Waters system (binary gradient module 2525, in-line degasser, sample manager 2767, photodiode array detector 2996) with a binary system for the solvent gradient. In particular, the eluent was a gradient of (99.9% water/0.1% HCOOH) and (99.9% MeCN/0.1% HCOOH) or (99.9% water/0.1% HCOOH) and (99.9% MeOH/0.1% HCOOH). Each compound was deposited on a 100-4.6 mm (5 mm) Zorbax SB-C.sub.18 column equilibrated with H.sub.2O/MeCN or H.sub.2O/MeOH 95:5.

[0095] This system was for example coupled to a Waters Micromass ZQ system with a ZQ2000 quadrupole analyser. The ionisation was performed by electrospray and the other parameters were as follows: source temperature 120° C., cone voltage 20 V, and continuous sample injection at a flow rate of 0.3 mL per min. The mass spectra were recorded in positive and negative ion mode in the range m/z 100-2000 and processed with Mass Lynx 4.0 software.

[0096] The infrared spectra were in particular recorded on a Spectrum Two equipped with a UATR Two (Perkin Elmer), Diamond/ZnSe (1 reflection).

[0097] NMR analyses were in particular performed on a Bruker Avance 400 Ultrashield spectrometer. The 1H-NMR and 13C spectra were recorded at room temperature at 400 MHz and 100 MHz respectively, the samples were dissolved in DMSO-d6 or CDCl.sub.3 at a concentration of approximately 5 mM. The DMSO singlet signal was set at 2.50 ppm. The chemical shifts are given in ppm and the coupling constants in Hz. The spectral data are consistent with the associated structures.

Preparation of the Compound 1

Synthesis of the Nitro Precursor 1′

[0098] ##STR00015##

TABLE-US-00001 M n m V Reagents eq. (g/mol) d (mmol) (mg) (mL) 5-fluoro-N-methylisatoic 1 195.15 / 1 195 / anhydride 2-nitroaniline 1 138.13 / 1 138 / 5-(2-methyl-1,3-thiazol-4-yl)- 1 209.00 / 1 209 / 2-thiophenecarbaldehyde Acetic acid / / / / / 2

[0099] The mixture was stirred at 120° C. for 3 hours under microwave irradiation. The residue was dissolved in EtOAc and washed successively with a saturated solution of NaHCO.sub.3 (three times), brine (twice) and water (once). The organic phase was dried on MgSO.sub.4, filtered and concentrated. The residue was purified by chromatography (solid deposition), eluted by a mixture of cyclohexane/EtOAc: 9/1->1/1 (yield: 62%).

Synthesis of the Compound 1

[0100] ##STR00016##

TABLE-US-00002 M n m V Reagent eq. (g/mol) (mmol) (mg) (mL) 1′ 1 480.07 0.44 273 / Zn 30 65.39 13.2 860 / EtOH / / / / 10 H.sub.2O / / / / 1.8 AcOH / / / / 0.9

[0101] The mixture was stirred overnight at room temperature, protected from light.

[0102] The residue obtained was purified by chromatography (solid deposit), eluted by a mixture of cyclohexane/EtOAc: 95/5->0/1, then re-precipitated by dissolving in the minimum of EtOAc and adding pentane (yield: 63%).

[0103] .sup.1H NMR (CDCl.sub.3): 2.69 (s, 3H); 2.91 (s, 3H); 4 (s broad, 2H); 5.76 (s, 1H); 6.54 (dd, 1H, J: 4, J: 8.9); 6.60 (td, 1H, J: 1.3, J: 7.8); 6.66 (d, 1H, J: 3.7); 6.75 (dd, 1H, J: 1.2, J: 8); 6.84 (dd, 1H, J: 1.4, J: 7.9); 7-7.1 (m, 3H); 7.1 (dd, 1H, J: 3.1, J: 8.7); 7.72 (dd, 1H, J: 3, J: 8.6).

[0104] .sup.13C NMR (CDCl.sub.3): 19.1; 36; 76.2; 111.9; 114.4 (d, J: 7.1), 115.5 (d, J: 24); 116.7; 118.8 (d, J: 7.4); 118.9; 121.3 (d, J: 23.1); 122.7; 125.2; 128.3; 129.2; 129.5; 137.8; 138.6; 142; 143.1; 148.7; 156.6 (d, J: 239); 161; 166.4.

[0105] LC/MS: retention time: 3.23 min.

[0106] M+H.: 451.4.

Preparation of the Compound 2

[0107] ##STR00017##

TABLE-US-00003 M n m V Reagent eq. (g/mol) (mmol) (mg) (mL) 5-fluoro-N-methylisatoic 1 195.03 1 195 / anhydride 2-aminophenol 1 109.13 1 109 / 5-(2-methyl-1,3-thiazol-4-yl)- 1 209.00 1 209 / 2-thiophenecarbaldehyde Acetic acid / / / / 2

[0108] The mixture was stirred at 130° C. for 2 hours. The residue was dissolved in EtOAc and washed successively with a saturated solution of NaHCO.sub.3 (three times), brine (twice) and water (once). The organic phase was dried on MgSO.sub.4, filtered and concentrated. The residue was purified by chromatography (solid deposition), eluted by a mixture of cyclohexane/EtOAc: 95/5->1/1, then re-precipitated by dissolving in minimum EtOAc and adding pentane (yield: 25%).

[0109] .sup.1H NMR (DMSO-d6): 2.62 (s, 3H); 2.9 (s, 3H); 6.16 (s, 1H); 6.75 (td, 1H, J: 1.3, J: 7.7); 6.79 (dd, 1H, J: 4, J: 9); 6.93-7.01 (m, 3H); 7.15 (td, 1H, J: 1.7, J: 7.5); 7.28 (d, 1H, J: 3.6); 7.35 (td, 1H, J: 3.2, J: 8.8); 7.58 (dd, 1H, J: 3.1, J: 8.8); 7.71 (s, 1H).

[0110] .sup.13C NMR (DMSO-d6): 18.5; 35.5; 75.3; 112.7; 113.7 (d, J: 23.6); 115 (d, J: 7); 116.7; 117.8 (d, J: 7); 118.8; 121.1 (d, J: 23.1); 122.6; 126.2; 128.3; 128.7; 129.9; 137.6; 139; 143.3; 147.8; 152.6; 155.3 (d, J: 235); 159.5; 165.9

[0111] IR: 1646; 1496; 1450; 1163; 806; 740

[0112] LC/MS: retention time: 3.24 min.

[0113] M+H.: 452.4.

Preparation of the Compound 3

[0114] ##STR00018##

TABLE-US-00004 Molec- Vol- ular Mass ume n Reagents Formula Mass (g) (mL) (mmol) N-methyl-5-fluoroisatoic C6H4FNO3 195.15 0.195 / 1.0 anhydride 3-aminophenol C6H7NO 109.13 0.109 / 1.0 5-(2-methyl-1,3-thiazol- C9H7NOS2 209.28 0.209 / 1.0 4-yl)-2- thiophenecarbaldehyde Acetic acid C2H4O2 60.05 / 2.00 /

[0115] The mixture was stirred at 130° C. for 2 hours. The residue was dissolved in EtOAc and the organic phase washed successively with a saturated NaHCO.sub.3 solution and distilled water. The organic phase was dried on MgSO.sub.4, filtered and concentrated under vacuum. The resulting brown solid was washed with a small amount of hot EtOAc to give a yellow powder (17%).

[0116] .sup.1H NMR (DMSO-d6): 2.62 (s, 3H); 2.95 (s, 3H); 6.43 (s, 1H); 6.7-6.78 (m, 3H); 6.81 (dd, 1H, J: 4.2, J: 9); 6.97 (d, 1H, J: 3.7); 7.20 (t, 1H, J: 8); 7.33 (d, 1H, J: 3.6); 7.36 (dd, 1H, J: 3, J: 8.8); 7.61 (dd, 1H, J: 3, J: 8.8); 7.71 (s, 1H).

[0117] .sup.13C NMR (DMSO-d6): 18.5; 35.4; 76; 112.8; 113.7; 113.7 (d, J: 23); 114; 115.4 (d, J: 7); 116.8; 118 (d, J: 7); 121.3 (d, J: 23); 122.9; 128.2; 129.5; 137.7; 138.7; 140.8; 143.2; 147.7; 155.5 (d, J: 235); 157.6; 159.8; 166.

[0118] IR: 3161 (broad); 1612; 1499; 1452; 1187; 1156; 795; 766; 730.

[0119] LC/MS: retention time=3.16 min.

[0120] M+H.: 452.3.

[0121] The compound 7, of the following formula, is prepared analogously to compound 2 or 3.

##STR00019##

Preparation of the Compound 4

Synthesis of the Nitro Precursor 4′

[0122] ##STR00020##

TABLE-US-00005 M n m V Reagents eq. (g/mol) (mmol) (mg) (mL) N-methylisatoic anhydride 1 177.04 1 177 / 2-nitroaniline 1 138.13 1 138 / 5-(2-methyl-1,3-thiazol-4-yl)- 1 209.00 1 209 / 2-thiophenecarbaldehyde Acetic acid / / / / 2

[0123] The mixture was stirred at 120° C. for 3 hours under microwave irradiation. The residue was dissolved in EtOAc and washed successively with a saturated solution of NaHCO.sub.3 (three times), brine (twice) and water (once). The organic phase was dried on MgSO.sub.4, filtered and concentrated under vacuum. The residue was purified by chromatography (solid deposition), eluted by a mixture of cyclohexane/EtOAc: 9/1->1/1 (yield: 49%).

Synthesis of the Compound 4

[0124] ##STR00021##

TABLE-US-00006 M n m V Reagents eq. (g/mol) (mmol) (mg) (mL) 4′ 1 462.08 0.79 365 / Zn 30 65.39 30 1500 / EtOH / / / / 10 H.sub.2O / / / / 1.8 AcOH / / / / 0.9

[0125] The mixture was stirred overnight at room temperature, protected from light.

[0126] The residue obtained was purified by chromatography (solid deposit), eluted by a mixture of cyclohexane/EtOAc: 95/5->0/1, then re-precipitated by dissolving in the minimum of EtOAc and adding pentane (yield: 81%).

[0127] .sup.1H NMR (DMSO-d6): 2.62 (s, 3H); 2.90 (s, 3H); 5.04 (s, 2H); 5.97 (s, 1H); 6.48 (td, 1H, J: 1.2, J: 7.3); 6.68 (dd, 1H, J: 1.2, J: 7.7); 6.77 (d, 1H, J: 8.2); 6; 6.87 (dd, 1H, J: 1, J: 8); 6.9-6.96 (m, 2H); 7.03 (td, 1H, J: 1.3, J: 8.4); 7.3 (d, 1H, J: 3.6); 7.48 (td, 1H, J: 1.5, J: 8.6); 7.72 (s, 1H); 7.88 (dd, 1H, J: 1.5, J: 7.7).

[0128] .sup.13C NMR (DMSO-d6): 18.5; 34.9; 74.5; 112.7; 113; 115.9; 116.1; 117; 118.3; 122.6; 124.2; 128.1; 128.4; 128.7; 129.4; 134.2; 137.6; 138.7; 143.8; 143.8; 146.7; 147.8; 160.9; 165.9

[0129] LC/MS: retention time=3.18 min.

[0130] M+H.: 433.4.

Preparation of the Compound 5

[0131] ##STR00022##

TABLE-US-00007 M n m V Reagents eq. (g/mol) (mmol) (mg) (mL) 2-aminobenzenesulfonamide 1 172.20 0.4 69 / 6-fluoro-N-methylisatoic 1 195.03 0.4 78 / anhydride 5-(2-methyl-1,3-thiazol-4-yl)- 1 209.28 0.4 84 / 2-thiophenecarbaldehyde Acetic acid / / / / 1

[0132] The mixture was stirred at 120° C. for 2 hours under microwave irradiation. The residue was dissolved in dichloromethane and washed successively with saturated NaHCO.sub.3 solution (three times), brine (twice) and water (once). The organic phase was dried on MgSO.sub.4, filtered and concentrated under vacuum. The residue was purified by chromatography (solid deposition), eluted by a mixture of cyclohexane/EtOAc: 9/1->0/1, then recrystallised in the dichloromethane (yield: 13%).

[0133] .sup.1H NMR (DMSO-d6): 2.63 (s, 3H); 2.85 (s, 3H); 6.18 (s, 1H); 6.85 (dd, 1H, J: 4.2, J: 9.1)]; 6.89 (dd, 1H, J: 1.3, J: 7.4); 6.91 (d, 1H, J: 3.6); 7.32 (d, 1H, J: 7.6); 7.39 (td, 1H, J: 3.2, J: 8.7); 7.47 (s, 2H); 7.57-7.59 (m, 3H); 7.78 (s, 1H); 8.02 (dd, 1H, J: 1.9, J: 7.5).

[0134] .sup.13C NMR (DMSO-d6): 18.5; 35.24; 76.7; 113.09; 113.7 (J: 23.6) 114.9 (J: 7); 116.6 (J: 7); 121.6 (J: 22.9); 122.6; 128.3; 128.84; 129.4; 132.1; 132.5; 136.1; 137.6; 138; 141.2; 143.3; 147.7; 155.1 (J: 235); 160.4; 166.

[0135] IR: 3175; 1653; 1496; 1474; 1450; 1357; 1342; 1160; 821; 810; 710.

[0136] LC/MS: retention time=3.20 min.

[0137] M+H.: 515.4.

Preparation of the Compound 6 (Out-of-Invention)

[0138] ##STR00023##

TABLE-US-00008 Molec- Vol- ular Mass ume n Reagents Formula Mass (g) (mL) (mmol) N-methyl-5-fluoroisatoic C6H4FNO3 195.15 0.180 / 0.92 anhydride 4-aminophenol C6H7NO 109.13 0.100 / 0.92 5-(2-methyl-1,3-thiazol- C9H7NOS2 209.28 0.192 / 0.92 4-yl)-2- thiophenecarbaldehyde Acetic acid C2H4O2 60.05 / 2.00 /

[0139] The mixture was stirred at 130° C. for 2 hours under microwave irradiation. The residue was dissolved in ethyl acetate and the organic phase was washed successively with a saturated solution of NaHCO.sub.3 and distilled water. The organic phase was dried on MgSO.sub.4, filtered and concentrated under vacuum. The resulting brown solid was washed with a small amount of hot EtOAc to give a yellow powder (73%).

[0140] .sup.1H NMR (DMSO-d6): 2.60 (s, 3H); 2.89 (s, 3H); 6.32 (s, 1H); 6.74-6.77 (m, 3H); 6.92 (wide s, 1H); 7.06 (d, 2H, J: 8.1); 7.3-7.35 (m, 2H); 7.57 (d, 1H, J: 6.6); 7.72 (s, 1H); 9.59 (s, 1H).

[0141] .sup.13C NMR (DMSO-d6): 18.5; 35.3; 76.4; 112.8; 113.7 (J: 24); 115.2 (J: 7); 115.4; 117.9 (J: 7); 121.2 (J: 23); 122.8; 128; 128.4; 131; 137.7; 138.5; 143.3; 147.7; 155.5 (J: 235); 156.3; 159.9; 166.

[0142] IR: 3216 (broad); 1630; 1513; 1498; 1267; 1163; 888; 811; 737; 711.

[0143] LC/MS: retention time=3.33 min.

[0144] M+H.: 452.4.

II. Measurement to Evaluate the Protective Activity of the Compounds of the Invention Against the Shiga Toxin

[0145] Protocol and Calculation of the IC.sub.50

[0146] The compounds were tested in either A549 cells (human lung epithelial cells) or HeLa cells (human uterine cancer cells) against the Shiga toxin (Stx-1 and/or Stx-2). The human cells are grown at 37° C. in an atmosphere containing 5% CO.sub.2 on 150 cm.sup.2 culture flasks in DMEM (Dulbecco's Modified Eagle Medium) containing 100 U/mL of penicillin and 100 μg/mL of streptomycin. The cells are seeded at a density of 50,000 cells per well in plates of 96 Cytostar-T solid scintillation bottom wells (FIG. 2). The cells (100 μL in complete DMEM:DMEM+10% fetal calf serum, SVF) are pre-incubated or not with the inhibitors (50 μL; different concentrations, 3 h pre-incubation). Complete medium supplemented with toxin (50 μL, variable concentration range) is then added to each well. After a 20 h incubation, the medium (200 μL) is removed and replaced with leucine-free DMEM medium (Eurobio) containing 10% SVF and 0.5 μCi/mL of .sup.14C-leucine (GE). After a 7 h incubation at 37° C., the incorporation of radioactivity by the cells is determined by reading the plates with a Wallac 1450 Microbeta trilux scintillation counter (PE).

[0147] As these toxins block the protein synthesis, the affected cells are no longer able to incorporate the radiolabelled leucine. In contrast, the cells treated with inhibitors still synthesise proteins and therefore incorporate the radiolabelled amino acid. As the cells concentrate the radioelement sufficiently close to the bottom of the well, this causes excitation of the scintillant contained in the plates and leads to photon emission detected by the scintillation counter (measured in counts per minute, cpm). This data is then expressed as a percentage of protein synthesis by the cells. The cytotoxicity curves can thus be plotted (FIG. 2) without inhibitor (white circles) or in the presence of an inhibitor (black circles). The analysis of the data by non-linear regression allows the EC.sub.50 to be estimated, i.e. the effective concentration for which 50% uptake of radioactive leucine is observed, which corresponds to 50% of viable cells. The higher the EC.sub.50 value, the greater the cell protection, as a higher concentration of toxin is required to generate the same cytotoxicity. It is thus possible to determine the effectiveness of the inhibitors by calculating the ratio R of the EC.sub.50 (FIG. 2). The higher the value (>1), the greater the cell protection.

[0148] Six cytotoxicity curves are obtained in the absence and then in the presence of increasing concentrations of inhibitor. For each concentration (C) of inhibitor, a percentage of protection is determined from the value of R calculated by the Prism software with Rmax corresponding to the maximum value of R in the series:

% protection=[(R−1)/(Rmax−1)]*100

[0149] The IC.sub.50 is then calculated, using the Prism software by non-linear regression, from a graph where the corresponding percentage of cell protection is plotted for each compound concentration (see FIG. 3).

[0150] IC.sub.50 corresponds to the concentration of compound giving 50% of its inhibiting power. The lower this concentration, the more powerful the inhibitor.

[0151] Results

TABLE-US-00009 Compound IC.sub.50(nM) Retro-2.1 (Control) 100.0 4 78.6 1 14.2 2 20.0 3 55.0 5 35.0 7 41.0 6 (out-of-invention) 483.0

[0152] The compound Retro-2.1 has the following formula:

##STR00024##

Conclusion

[0153] These results show that the tested compounds are all more potent inhibitors of the cellular effect of the Shiga toxin than the compound Retro-2.1 (out-of-invention control, in which the dihydroquinazolinone carries an unsubstituted phenyl), as well as the compound 6 (out-of-invention, in which the dihydroquinazolinone carries a phenyl substituted in the para position by a hydroxyl group).

III. Measure to Evaluate the Protective Activity of the Compounds of the Invention Against the Ricin

[0154] The protocol and the calculation of the IC.sub.50 of the compounds of the invention is similar to that presented in paragraph II, taking the ricin as toxin.

IV. Measurements to Evaluate the Protective Activity of the Compounds of the Invention Against Viruses Using Retrograde and/or Syntaxin 5-Dependent Transport, in Particular Viruses Entering the Cells by Endocytosis

[0155] Cells as indicated below were seeded at a density of 50% in plates of 96 wells in a MEM medium supplemented with 10% of fetal bovine serum and incubated at 37° C., 5% of CO.sub.2. The cells were allowed to stand overnight. The test compounds were dissolved in the DMSO at 20 mM each. The next day, each compound was diluted 100 times in MEM with serum, serially diluted (to 5 concentrations) and added in equal volume to the cell medium in each well. After 1 hour of pre-incubation, the virus was added at a multiplicity of infection (MOI) as indicated below. The viruses used comprise in particular recombinant viruses that encode a green fluorescent protein (Towner et al. 2005. Virology 332:20-7; or ANCHOR viruses supplied by NEOVIRTECH). These viruses are capable of replication and exhibit a normal pathogenesis in animals. The cells were then fixed with 10% formalin for the time indicated below. The cell nuclei were stained with DAPI using standard methods. The infection was then analysed by photographing each well using an epifluorescent microscope. The total and infected cells were counted by counting DAPI-stained cells and green fluorescent cells respectively using Cell Profiler software. The fraction of infected cells was calculated by dividing the number of green fluorescent cells by the total number of cells. The evaluation of the IC.sub.50 was performed as a function of the cell number using Combenefit software (Di Veroli et al. Bioinformatics 2016, 32(18), 2866-8).

[0156] Infection and fixation conditions: [0157] MRC5 cells with hCMV-ANCHOR, MOI 1; fixation at 6 days; [0158] HeLa cells with VACV-ANCHOR, MOI 0.1; fixation at 48 hours; [0159] RK13 cells with Myxomavirus T1-ANCHOR, MOI 0.05; fixation at 6 days; [0160] Hep2 cells with RSV; MOI 0.2; fixation at 72 hours.

[0161] Toxicity

[0162] Compound concentrations of the invention from 100 μM to 0.2 μM were tested (twice). The cells as described above were fixed 72 hours after addition of the compounds of the invention. The number of cells counted per well is normalized to the number of cells counted in the corresponding wells treated with DMSO only. The evaluation of the CC.sub.50 was performed as a function of cell number using the Combenefit software mentioned above.

[0163] Results

[0164] The results obtained are reported in the following table:

TABLE-US-00010 6 (out-of- Retro-2.1 2 1 invention) HeLa/ CC50 (nM) >50,000 32,300 >50,000 20,400 VacV CI50 (nM) 37.8 3.61 1.05 628 Selectivity 1,323 8,947 47,619 32 index RK13/ CC50 (nM) 3,350 ND 7,460 12,900 MyxV CI50 (nM) 74.5 14.2 1.44 5,800 T1 Selectivity 45 ND 5,181 2 index MRC5/ CC50 (nM) >50,000 21,000 21,500 16,700 hCMV CI50 (nM) 11.6 <1 <1 281 Selectivity 4,310 21,000 21,500 59 index Hep2/ CC50 (nM) >50,000 37,600 >50,000 25,600 RSV CI50 (nM) 162 20 ND 2,200 Selectivity 309 1,880 ND 12 index

V. Measure to Evaluate the Protective Activity of the Compounds of the Invention Against a Bacteria Using the Retrograde and/or Syntaxin 5-Dependent Transport, in Particular a Bacteria Entering the Cells by Endocytosis

[0165] HeLa229 cells are pre-treated with the compound of the invention to be evaluated at concentrations of 25.50 and 75 μM for 30 minutes until the bacteria is added to the cells.

[0166] The cells showing a primary infection are lysed 24 hours after the infection (hpi).

[0167] Cells treated with said compound are also lysed 48 h after the infection and the resulting lysate is used to infect new HeLa229 cells, which are lysed 24 h after the infection and analysed with the primary infection samples by immunoblot. The growth of the bacteria is detected using ad hoc antibodies and the actin is used as a control.

VI. Synthesis of Prodrugs According to the Invention

[0168] Prodrugs according to the present invention have been synthesized as follows:

##STR00025## ##STR00026## ##STR00027##

[0169] The compounds thus obtained have an excellent solubility in water (>700 mg/mL, possible administration in minipumps, infusion, i.v. injection). Their cleavage by the plasma amidases and/or esterases is rapid (e.g. with a t.sub.1/2 of 2.6 hours in mouse plasma at 37° C.).

[0170] Other prodrugs, in the form of thermogelling polymers, have been obtained as follows:

##STR00028##

[0171] Or as follows:

[0172] (Compound 2)-PLGA1036-PEG1450-PLGA1036-(Compound 2) (Mn=4,393 g/mol) or

[0173] (Compound 2)-C(═O)—(CH.sub.2).sub.2—C(═O)—PLGA1036-PEG1450-PLGA1036-C(═O)—(CH.sub.2).sub.2—C(═O)-(Compound 2), in particular of the following formula:

##STR00029##

[0174] soluble in water and with thermogelation properties.

[0175] The PLGA1036-PEG1450-PLGA1036 is for example obtained as follows:

##STR00030##

[0176] The poly(ethylene glycol) 1450 (3.00 g -2.07 mmol) is placed in a schlenk, heated to 100° C. in an oil bath. The schlenk is placed under vacuum for 2 h with stirring. The glycolide (0.6 g-5.13 mmol) and D,L-Lactide (2.64 g-18.3 mmol) were added to the schlenk and the mixture was heated to 130° C. until the solids were completely melted. The Sn(Oct).sub.2 (one drop) is added. The mixture is heated for 20 hours at 130° C. with stirring, under nitrogen. The mixture was brought to RT and dissolved in 20 mL of acetone. The solution is added drop by drop to distilled water at 4° C. with stirring. The mixture was stirred at 4° C. until the polymer was completely solubilised and then the mixture was heated to 80° C. The polymer is recovered by decantation. This sequence is repeated then the solid is dissolved in dichloromethane. The organic phase is dried on MgSO.sub.4, filtered and evaporated to give a translucent gum which was dried under vacuum at 40° C. for 15 hours.

[0177] Purified product: 3.8 g (49%).

[0178] Molar mass (PLA)=1,707 g/mol; Molar mass (PGA)=365 g/mol, corresponding to a compound PLGA1036-PEG1450-PLGA1036 (Mn=3,522 g/mol; LA/GA=3.8).