Dihydroquinazolinones exhibiting improved protective activity against intracellularly acting toxins and intracellular viruses and bacteria

Abstract

The present invention relates to a compound of general formula (I) and also the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts thereof, and also to the use thereof for the prevention and / or treatment of disorders caused by intracellularly acting toxins using retrograde transport, or by viruses or bacteria using retrograde and / or syntaxin 5-dependent transport to infect cells.

Description

[0001] Description

[0002] Title: DIHYDROQUINAZOLINONES WITH ENHANCED PROTECTIVE ACTIVITY AGAINST TOXINS WITH AN INTRACELLULAR MODE OF ACTION, VIRUSES AND INTRACELLULAR BACTERIA

[0003] The present invention relates to a novel family of compounds of the 2,3-dihydroquinazolin-4(lH)-one type and their use as inhibitors of the toxic effects of toxins with intracellular activity, such as ricin, abrin and Shiga toxins, using retrograde transport to intoxicate cells, or viruses or bacteria using retrograde and / or syntaxin 5-dependent transport to infect cells, in particular viruses or bacteria entering cells by endocytosis, or intracellular parasites.

[0004] Technical field

[0005] Intracellularly active toxins that utilize retrograde transport represent a major public health risk, some of which are associated with more than one million deaths worldwide each year. These toxins include ricin (produced in the seeds of the plant Ricinus communis), abrin (produced in the seeds of the plant Abrus precatorius), Shiga toxin and Shiga-like toxins (Stxs) produced by Shigella dysenteriae (Stx) and E. coli (Stxl and Stx2), pertussis toxin (Bordelella pertussis, the agent of whooping cough), subtilase cytotoxin, and heat-labile enterotoxin (E. coli).

[0006] Ricin is a 66 kDa glycoprotein composed of two polypeptide chains linked by a disulfide bridge. The B chain (RTB, 262-residue lectin) allows the toxin to bind to glycolipids and glycoproteins in cell membranes and ensure its entry into the cell. The A chain (RTA, 267 amino acids) ensures the N-glycosidase enzymatic function of ricin, catalyzing the removal of adenine 4324 from the 28S RNA of target cells and causing the cessation of protein synthesis.

[0007] Abrin is an extremely toxic toxalbumin present in the seeds (EAbrus precatorius) with a structure and mechanism of action similar to ricin.

[0008] Shiga toxins include Shiga toxin (Stx) produced by Shigella dysenteriae and Shiga-like toxins 1 (Stxl) and 2 (Stx2) produced by enterohemorrhagic strains of E. coli. Stx and Stxl toxins are 99% identical, whereas Stxl and Stx2 share only 56% amino acid sequence identity. The toxin A subunit (StxA) carries the same enzymatic activity as ricin and identically targets 28S RNA. The pentameric B subunit (StxB) allows the toxin to bind to the cell via its interaction with the globo-triaosylceramide Gb3, which ensures its internalization and intracellular routing.

[0009] After binding to its membrane receptors, ricin is internalized in these cells by multiple endocytic pathways to reach the trans-Golgi network where it is transported to the endoplasmic reticulum (ER) by retrograde transport (Johannes, L. et al., Cell 2008, 135, 1175-87). Abrin acts by penetrating the cell by binding to the carbohydrate chains of glycoproteins and glycolipids on the cell surface before being internalized inside it. This process, little known, is similar in terms of structure and mechanism of action to that of ricin. Shiga toxins are internalized by a unique endocytic pathway and also reach the Golgi apparatus and then the endoplasmic reticulum (ibid.). The toxins are then partially unfolded and the A chain / subunit is translocated into the cytosol (Lord, JM et al., Biochemical Society Transactions 2003, 31, 1260).The final stage of the action of these toxins therefore takes place in the cytoplasm of the cells, the toxins bind to the ribosomes with great efficiency and cleave adenine 4324 of the 28S RNA of the 60S subunit of the ribosome. This depurination of the 28S RNA causes the cessation of protein synthesis and leads to cell death.

[0010] To counter the threat posed by these toxins, several types of antitoxins have been developed: neutralizing antibodies, inhibitors of enzymatic activity (small molecules, substrate analogs), soluble receptor mimics, and chemical compounds acting on the cells targeted by the toxin.

[0011] In recent years, the search for new molecules aimed at blocking the intracellular routing of intracellularly active toxins has accelerated. The main advantage of this type of molecule is its broad-spectrum activity, as these molecules can effectively protect cells against various toxins that use the retrograde pathway.

[0012] Poxviruses are DNA viruses related to the Poxviridae family, infecting animals and causing diseases such as monkeypox, smallpox, vaccinia, cowpox, camelpox affecting camelids (camals, dromedaries, llamas) and molluscum contagiosum. Poxviruses with animal reservoirs, particularly monkeypox and camelpox, are emerging diseases with a risk of spread due to the increase in international transport, the fashion for pets, especially exotic animals, the proximity of humans to their working animals, and the lack of smallpox vaccine protection. There is one anti-smallpox molecule on the market, ST246-tecovirimat or TPOXX®.However, monotherapy with this molecule generates resistant strains of virus and there is a consensus on the need to have several drugs against poxviruses, particularly in a OneHealth approach.

[0013] There therefore remains a strong need to offer effective treatments against this family of viruses.

[0014] The present invention specifically seeks to provide molecules that can protect cells against toxins with intracellular activity, against viruses or bacteria and intracellular parasites.

[0015] According to a first embodiment, the present invention relates to a compound of general formula (I):

[0016] - p is equal to 1, 2 or 3;

[0017] - R 1represents at each occurrence, independently, a hydrogen atom, a halogen atom, an alkoxy radical of 1 to 3 carbon atoms, in particular a methoxy group, -NO2, or -NH2;

[0018] - R 2 and R 3 independently represent a group chosen from -OH, -OCH3, -SH, -SCH3, -OR 4 , -NH2, -NHR 5 , -NR 7 R 8 , -SO2-NH2, -SO2-NH-R 6 , SO3H, or a halogen atom;

[0019] - R 4 , R 5 and R 6 independently represent a group of formula (II) - L-(X)i-(PEG)-(Y)jZ, where:

[0020] - i and j independently represent 0 or 1; - L represents -C(=0)- or -C(=O)-(CH2)kC(=O)-, with k being equal to 1, 2 or 3, in particular 2;

[0021] - R 7 and R 8 represent, independently of one another, a linear or branched C1 to C3 alkyl;

[0022] - X and Y independently represent a poly(lactic acid) or a poly(lactic acid-co-glycolic acid);

[0023] - PEG represents a poly(ethylene glycol);

[0024] - Z represents a group selected from H, C1-C3 alkyl, -OH, O-C1-C3 alkyl, or -L-Re, where L is defined as previously and Re is a residue of formula (I) linked to said group -L-(X)i-(PEG)-(Y)j- defined previously via its group R 2 or R 3 , said group R 2 or R 3 being -OH, -NH2 or -SO2-NH2;

[0025] - R 9 represents -CH3 or -CH2-OH, in particular represents CH3. as well as stereoisomeric forms, mixtures of stereoisomeric forms or their pharmaceutically acceptable salts.

[0026] According to another embodiment, the present invention relates to a compound of general formula (!):

[0027] (From where

[0028] - p is equal to 1, 2 or 3;

[0029] - R 1 represents at each occurrence, independently, a hydrogen atom, a halogen atom, an alkoxy radical of 1 to 3 carbon atoms, in particular a methoxy group, -NO2, or -NH2;

[0030] - R 2 and R 3 independently represent a group chosen from -OH, -OCH3, -SH, -SCH3, -OR 4 , -NH2, -NHR 5 , -NR 7 R 8 , -SO2-NH2, -SO2-NH-R 6 , SO3H, or a halogen atom; - R 4 , R 5 and R 6 independently represent a group of formula (II) - L-(X)i-(PEG)-(Y)jZ, where:

[0031] - i and j independently represent 0 or 1;

[0032] - L represents -C(=O)- or -C(=O)-(CÜ2)kC(=O)-, with k being equal to 1, 2 or 3, in particular 2;

[0033] - R 7 and R 8represent, independently of one another, a linear or branched C1 to C3 alkyl;

[0034] - X and Y independently represent a poly(lactic acid) or a poly(lactic acid-co-glycolic acid);

[0035] - PEG represents a poly(ethylene glycol);

[0036] - Z represents a group selected from H, C1-C3 alkyl, -OH, O-C1-C3 alkyl, or -L-Re, where L is defined as previously and Re is a residue of formula (I) linked to said group -L-(X)i-(PEG)-(Y)j- defined previously via its group R 2 or R 3 , said group R 2 or R 3 being -OH, -NH2 or -SO2-NH2; as well as stereoisomeric forms, mixtures of stereoisomeric forms or their pharmaceutically acceptable salts.

[0037] Particularly surprisingly, this new family of molecules exhibits biological activity as effective or even superior to the compounds of the family of molecules described in international application WO2020 / 109510 A1. Structurally very close, these molecules differ from those of the present invention by the presence of one or two ortho and meta substitutions in the phenyl ring.

[0038] Furthermore, the molecules of the present invention exhibit a better calculated solubility (see the “log S” column in Table 3 below) which, for certain molecules only, unexpectedly results in a better bioavailability compared to the molecules known in the art, and in particular compared to the compounds A to D presented in Tables 2 and 3 below. These properties are notably increased when the molecules are administered in a vehicle solution as described below. Indeed, the poor solubility of the Retro-2.1 compound and its analogues in “conventional” administration vehicles poses a major problem for formulators, in particular when it comes to producing pharmaceutical compositions on a large scale.According to a second aspect, the invention relates to a compound of general formula (I) as described above, for its use for the prevention and / or treatment of disorders induced by toxins with an intracellular mode of action using retrograde transport, or by viruses or bacteria using retrograde transport and / or dependent on syntaxin 5 to infect cells, in particular viruses or bacteria entering cells by endocytosis, or by intracellular parasites.

[0039] The invention also relates to a pharmaceutical composition or medicament comprising at least one compound of general formula (I) as defined above as active ingredient, and a pharmaceutically acceptable vehicle, said pharmaceutical composition or said medicament being in particular suitable for administration by the air, oral, parenteral, local, intramuscular or subcutaneous routes.

[0040] Brief description of the drawings

[0041] [Fig 1] represents the plasma concentration (in nM) of compound 1 (white circle), compound B (white square), compound C (white triangle) and compound E (white diamond) as a function of time (in hours) administered to female BALB / c mice orally at a concentration of 33 mg / kg in a Lipiodol © / castor oil mixture in mass proportions of 30% / 70%.

[0042] [Fig 2] represents the plasma concentration (in nM) of compound 1 (white circle), compound B (white square), compound C (white triangle) and compound E (white diamond) as a function of time (in hours) administered to female BALB / c mice subcutaneously at a concentration of 33 mg / kg in a Lipiodol © / castor oil mixture in mass proportions of 30% / 70%.

[0043] [Fig 3] represents the plasma concentration (in nM) of compound 1 in formulation B (white circle), compound 1 in formulation C (black circle), compound B in formulation B (white square) and compound B in formulation C (black square) as a function of time (in hours) administered subcutaneously in female BALB / C mice at a concentration of 33 mg / kg.

[0044] [Fig 4] represents a simulation of the plasma concentration (in nM) of compound B (left graph) or compound 1 (right graph) versus time (in hours) administered twice daily subcutaneously to female BALB / c mice at a concentration of 33 mg / kg in formulation C. [Fig 5] represents the residual percentage (in %) of the plasma concentration of compound 1 (curve 1), compound A (curve 2), compound B (curve 5), compound C (curve 4) and compound E (curve 3) versus time (in hours) incubated for 45 minutes with murine (A) or human (B) liver microsomes in the presence of NADPH versus time (in minutes).

[0045] In Figure 5A, the 30-hour curves are, from top to bottom, those of compound 1, compound A, compound E, compound C, and compound B.

[0046] In Figure 5B, the 30-hour curves are, from top to bottom, those of compound E, compound 1, compound B, and compound C.

[0047] [Fig 6] represents a simulation of the plasma concentration (in pM) of (S)-compound 1 as a function of time (in hours) administered twice daily subcutaneously (top graph) or once daily subcutaneously (bottom graph) in female BALB / c mice at a concentration of 33 mg / kg.

[0048] Statement of the invention

[0049] As part of its research on compounds blocking retrograde transport and more particularly studies on compounds more advantageous than those of the aforementioned work, the Applicant has identified a new family of compounds derived from 2,3-dihydroquinazolin-4(1H)-one, bearing a phenyl specifically substituted in di-ortho positions (ortho and ortho), which unexpectedly exhibit a biological activity superior to that of Retro-2.2, the most active of the molecules already described, and therefore a marked interest for the prevention and / or treatment of poisonings by at least one toxin with an intracellular mode of action using retrograde transport to infect mammalian eukaryotic cells, but also for the prevention and / or treatment of infections by viruses or bacteria using retrograde transport and / or dependent on syntaxin 5 to infect cells, in particular viruses or bacteria entering cells by endocytosis, or by intracellular parasites.

[0050] Thus, the present invention relates to a compound of general formula (I): Or

[0051] - p is equal to 1, 2 or 3;

[0052] - R 1 represents at each occurrence, independently, a hydrogen atom, a halogen atom, an alkoxy radical of 1 to 3 carbon atoms, in particular a methoxy group, -NO2, or -NH2;

[0053] - R 2 and R 3 independently represent a group chosen from -OH, -OCH3, -SH, -SCH3, -OR 4 , -NH2, NHR 5 , -NR 7 R 8 , -SO2-NH2, -SO2-NH-R 6 , SO3H, or a halogen atom;

[0054] - R 4 , R 5 and R 6 independently represent a group of formula (II) - L-(X)i-(PEG)-(Y)jZ, where:

[0055] - i and j independently represent 0 or 1;

[0056] - L represents -C(=O)- or -C(=O)-(CH2)kC(=O)-, with k being equal to 1, 2 or 3, in particular 2;

[0057] - R 7 and R 8 represent, independently of one another, a linear or branched C1 to C3 alkyl;

[0058] - X and Y independently represent a poly(lactic acid) or a poly(lactic acid-co-glycolic acid);

[0059] - PEG represents a poly(ethylene glycol);

[0060] - Z represents a group selected from H, C1-C3 alkyl, -OH, O-C1-C3 alkyl, or -L-Re, where L is defined as previously and Re is a residue of formula (I) linked to said group -L-(X)i-(PEG)-(Y)j- defined previously via its group R 2 or R 3 , said group R 2 or R 3 being -OH, -NH2 or -SO2-NH2;

[0061] - R 9 represents -CH3 or -CH2-OH, in particular represents CH3. as well as stereoisomeric forms, mixtures of stereoisomeric forms or their pharmaceutically acceptable salts.

[0062] According to one embodiment, R 9 represents - CH3. According to one embodiment, p is equal to 1.

[0063] According to one embodiment, R 1 represents a halogen atom, in particular a fluorine atom.

[0064] According to one embodiment, the invention relates to a compound of general formula (I) in which R 2 and R 3 independently represent a group chosen from -OH, -OCH3, -SH, -SCH3, -OR 4 , -NH2, -NHR 5 , -NR 7 R 8 , -SO2-NH2, -SO2-NH-R 6 , SO3H, in particular R 3 , represents a -OH group, R 4 , R 5 , R 6 , R 7 and R 8 being defined as previously.

[0065] According to one embodiment, the compound of general formula (I) according to the invention is of the following formula (Ia): where R 2 is as defined previously.

[0066] According to a particular embodiment, the compound of general formula (I) according to the invention is of the following general formula (Ib): in which R 2 and R 3 are chosen from the following combinations: R 2is an -OH group and R 3 is a -NH2, R group 2 is an -OH group and R 3 is a group -SO2NH2, R 2 is an -OH group and R 3 is a group -SH, R 2 is an -OH group and R 3 is a group -SO3H, R 2 is an -OH group and R 3 is a group -SMe, R 2 is an -OH group and R 3 is a group - OMe, R 2 is an -OH group and R 3 is a halogen atom, and in particular is a fluorine atom, R 2 is a -NH2 group and R 3 is a -NH2, R group 2 is a group - NH2 and R 3 is a group -SO2NH2, R 2 is a -NH2 group and R 3 is a group -SH, R 2 is a -NH2 group and R 3 is a group -SO3H, R 2 is a group - NH2 and R 3 is a group -SMe, R 2 is a group - NH2 and R 3 is a group -OMe, R 2 is a group - NH2 and R 3is a halogen atom, and in particular is a fluorine atom, R 2 is a group -SH and R 3 is a group -SH, R 2 is a group -SH and R 3 is a group -SO2NH2, R 2 is a group -SH and R 3 is a group -SO3H, R 2 is a group -SH and R 3 is a group -SMe, R 2 is a group -SH and R 3 is a group - OMe, R 2 is a group -SH and R 3 is a halogen atom and in particular is a fluorine atom, R 2 is a group -OMe and R 3 is a group -OMe, R 2 is a group -OMe and R 3 is a group -SMe, R 2 is a group -OMe and R 3 is a halogen atom, and in particular is a fluorine atom, R 2 is a group -SMe and R 3 is a group -SMe, R 2 is a group -SMe and R 3 is a halogen atom, and in particular is a fluorine atom, and R 2is a halogen atom and in particular is a fluorine atom and R 3 is a halogen atom and in particular is a fluorine atom, where Me is a methyl.

[0067] According to a particular embodiment, the compound has the following formula:

[0068] In the application, this compound will be referred to as “compound 1”.

[0069] According to a particular embodiment, the compound has the following formula:

[0070] This compound is a metabolite of compound 1. By "metabolite" is meant a transformed form of a compound, following in vivo or in vitro administration of said compound. For example, this transformation may consist of a hydroxylation of the compound. With regard to the group of formula (II) -L-(X)i-(PEG)-(Y)jZ, L represents in particular -C(=O)- when R 2 or R 3 represents -NHR 5 or -SO2-NH-R 6, and L represents in particular- C(=O)-(CH2)kC(=O)- when R 2 or R 3 represents -OR 4 .

[0071] According to one embodiment, the group of formula (II) -L-(X)i-(PEG)-(Y)jZ is chosen from -L-PEG, -L-PEG-PLA, and -L-PLGA-PEG-PLGA, with L representing in particular - C(=O)- when R 2 or R 3 represents -NHR 5 or -SO2-NH-R 6 , or in particular-C(=O)-(CH2)k- C(=O)- when R 2 or R 3 represents -OR 4 .

[0072] According to one embodiment, the group of formula (II) -L-(X)i-(PEG)-(Y)jZ is chosen from -C(=O)-(CH2-CH2-O) m -CH3, especially when R 2 or R 3 represents -NHR 5 or -SO2- NH-R 6 , and -C(=O)-(CH2)kC(=O)-(O-CH2-CH2)m-OCH3, especially when R 2 or R 3 represents -OR 4 , m being from 1 to 500, m being in particular from 30 to 60, more particularly from 45.

[0073] According to one embodiment, the compound of general formula (I) is of the following formula (III): the groups mentioned in formula (III) being as defined above.

[0074] According to one embodiment, the compound of general formula (I) according to the invention is chosen from:

[0075] and (Compound l)-PLGA1036-PEG1450-PLGA1036-(Compound 1) or

[0076] (Compound 1)-C(=0)-(CH2)2-C(=0)-PLGA1036-PEG1450-PLGA1036-C(=0)-(CH2)2- C(=O)-(Compound 1), in particular of the following formula:

[0077] The compounds of formula (I) may be in the form of an R-enantiomer, in the form of an S-enantiomer, or in the form of a racemic mixture. According to a particular embodiment, the compounds of formula (I) may be in the form of an S-enantiomer.

[0078] Certain compounds of general formula (I) constitute prodrugs. For illustrative purposes, mention may be made of compounds of general formula (I) for which R 4 , R 5 and R 6 independently represent a group of formula (II) -L-(X)i-(PEG)- (Y)jZ, where:

[0079] - i and j independently represent 0 or 1;

[0080] - L represents -C(=O)- or -C(=O)-(CH2)kC(=O)-, with k being equal to 1, 2 or 3, in particular 2.

[0081] Other compounds of general formula (I) can be coupled with given molecules to form prodrugs.

[0082] Prodrugs in particular make it possible to improve the bioavailability of compounds, and in particular to increase their selectivity for a targeted target by increasing their capacity to pass through cell membranes.

[0083] Coupling means the formation of a chemical bond between compounds of formula (I) and other molecules. These bonds can be covalent bonds, ionic or iono-covalent bonds, metallic bonds, hydrogen bonds, or Van der Waals forces.

[0084] According to a particular embodiment, certain compounds of general formula (I) can be coupled with hydrophilic molecules.

[0085] According to a particular embodiment, certain compounds of general formula (I) can be coupled with amphiphilic molecules.

[0086] According to a particular embodiment, certain compounds of general formula (I) can be coupled with lipophilic molecules.

[0087] In particular, certain compounds of general formula (I) may be coupled with fatty acids. Fatty acids suitable for such coupling are saturated fatty acids and unsaturated fatty acids. The following saturated fatty acids may be mentioned in particular: caprylic acid (8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0), behenic acid (22:0), lignoceric acid (24:0) and ceroic acid (26:0).

[0088] The following unsaturated fatty acids may also be mentioned: myristoleic acid (14:1), palmitoleic acid (16:1), sapienic acid (16:1), oleic acid (18:1), elaidic acid (18:1), trans-vaccenic acid (18:1), linoleic acid (18:2), linolelaidic acid (18:2), a-linolenic acid (18:3), y-linolenic acid (18:3), dihomo-y-linolenic acid (20:3), arachidonic acid (20:4), eicosapentaenoic acid (20:5), clupanodonic acid (22:5) and docosahexaenoic acid (22:6).

[0089] According to another aspect, the invention relates to a pharmaceutical composition or a medicament comprising at least one compound of general formula (I) as defined above, as active ingredient, and a pharmaceutically acceptable vehicle, said pharmaceutical composition or said medicament being in particular suitable for administration by the air, oral, parenteral, local, intramuscular or subcutaneous routes.

[0090] It should be noted that all the embodiments mentioned above in relation to the compound of general formula (I) also apply here, alone or in combination.

[0091] According to another aspect, the invention relates to a compound of general formula (I) as defined above, for its use for the prevention and / or treatment of disorders induced by toxins with an intracellular mode of action using retrograde transport, or by viruses or bacteria using retrograde transport and / or dependent on syntaxin 5 to infect cells, in particular viruses or bacteria entering cells by endocytosis, or by intracellular parasites.

[0092] It should be noted that all the embodiments mentioned above in relation to the compound of general formula (I) also apply here, alone or in combination.

[0093] According to one embodiment, the invention relates to a compound of general formula (I) as defined above, for its use for the prevention and / or treatment of disorders induced by toxins with an intracellular mode of action using retrograde transport. According to a particular embodiment, said toxins with an intracellular mode of action using retrograde transport are chosen from ricin, abrin, Shiga toxin and Shiga-like toxins (Stxs) produced by Shigella dysenteriae (Stx) and E. coli (Stxl and Stx2, Stx, Stx la, Stx2a, Stx2c, Stx2d, Stx2e as described for example by Melton-Celsa, Microbiol Spectr. 2014; 2(2)), pertussis toxin (Bordetella pertussis agent of whooping cough), subtilase cytotoxin and heat-labile enterotoxin (E. coli).

[0094] According to one embodiment, the invention relates to a compound of general formula (I) as defined above, for its use for the prevention and / or treatment of disorders induced by viruses or bacteria using retrograde transport and / or dependent on syntaxin 5 to infect cells, in particular viruses or bacteria entering cells by endocytosis, or intracellular parasites, such as leishmaniasis parasites.

[0095] According to a particular embodiment, the viruses are pox viruses, in particular smallpox virus, monkeypox virus, vaccinia virus and leporipoxviruses, in particular myxomatosis virus, adeno-associated viruses, in particular serotype 2, polyomaviruses, in particular polyomavirus JC and polyomavirus BK, papillomaviruses, filoviruses, in particular Ebola viruses and Marburg virus, enteroviruses, in particular enterovirus 71, herpesviruses, in particular Herpes simplex virus type 2 and cytomegalovirus (hCMV), viruses of the genus Arenavirus, in particular lymphocytic choriomeningitis virus, and pneumoviruses, in particular respiratory syncytial virus.

[0096] According to a particular embodiment, the bacteria are bacteria of the order Chlamydiales, in particular of the genus Chlamydia, such as Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psyttaci, or Symkania, such as Symkania negevensis.

[0097] According to a particular embodiment, the disorders induced by intracellular parasites are leishmaniasis, in particular induced by trypanosomes of the genus Leishmania, in particular Leishmania infantum, Leishmania donovani or Leishmania infantum / donovani hybrid.

[0098] The present invention also relates to a method for preventing and / or treating disorders induced by toxins with intracellular activity using retrograde transport and comprising administering to an individual in need thereof an effective amount of at least one compound of general formula (I) as defined above.

[0099] Finally, the present invention relates to the use of at least one compound of general formula (I) as defined above for the manufacture of a medicament for the treatment of disorders induced by toxins with intracellular activity using retrograde transport.

[0100] It should be noted that all the embodiments mentioned above in relation to the compound of general formula (I) also apply here, alone or in combination.

[0101] According to a particular embodiment, a compound of general formula (I) as defined above may be useful in the treatment of human diseases such as acute poisoning by ricin or abrin toxins; in the prevention of complications linked to hemolytic uremic syndrome; in the treatment of infections by poxviruses, in particular smallpox, monkeypox, cowpox, camelpox, or vaccinia infections; in the treatment of enterovirus infections.

[0102] According to a particular embodiment, a compound of general formula (I) as defined above may be useful in the treatment of animal diseases such as the treatment of myxomatosis in rabbits, monkeypox in monkeys or camelpox in camels.

[0103] Synthesis

[0104] The compounds of the present invention may be prepared according to methods well known to those skilled in the art, including, but not limited to, those described below, or by modifications of these methods by applying standard techniques known to those skilled in the art of organic synthesis. Suitable modifications and substitutions will be well known, readily apparent, or readily available from the scientific literature to those skilled in the art. In particular, such methods may be found in RC Larock, Comprehensive Organic Transformations, Wiley-VCH Publishers, 1999.

[0105] All methods disclosed within the scope of the present invention are contemplated to be practiced at any scale, including milligram, gram, multigram, kilogram, multikilogram, or commercial industrial scale. It is noted that the compounds of the present invention may comprise an asymmetrically substituted carbon atom, and may be isolated in optically active or racemic forms. Thus, all chiral, diastereomeric, racemic, isomeric forms of a structure are contemplated, unless the specific stereochemistry or isomeric form is specifically indicated. It is well known to prepare and isolate such optically active forms.For example, mixtures of stereoisomers can be separated by standard techniques, including, but not limited to, resolution of racemic forms, conventional, reversed-phase, and chiral chromatography, preferential salt formation, recrystallization, and others, or by chiral synthesis from chiral starting materials or by deliberate synthesis of target chiral centers.

[0106] The compounds of the present invention may be prepared by various synthetic routes. In the reactions described below, it may be necessary to protect reactive functional groups, for example, hydroxy, amino, or thio groups, when present in the final product, to prevent their participation in unwanted side reactions. Conventional protecting groups may be used in accordance with common practice, for example, see T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Chemistry, 3rd ed., John Wiley and Sons, 1999; J.F.W. McOmie in Protective Groups in Organic Chemistry, Plenum Press, 1973.

[0107] The reagents and starting compounds are commercially available, or readily synthesized by techniques well known to those skilled in the art. All substituents, unless otherwise indicated, are as defined above.

[0108] Compounds of general formula (I) can be obtained according to the following reaction:

[0109] This reaction can be carried out in an organic solvent.

[0110] According to one embodiment, the three starting compounds are mixed in an organic solvent, in particular acetic acid. According to one embodiment, the organic solvent is heated, in particular at reflux or under microwave irradiation, for example at a temperature of from 100°C to 180°C, in particular from 110°C to 140°C, more particularly from 120°C to 130°C.

[0111] The heating time is from 10 minutes to 6 hours, especially from 30 minutes to 5 hours, especially from 1 hour to 4 hours, especially from 2 to 3 hours.

[0112] R 1 and p are as defined above.

[0113] According to one embodiment, R 2 and R 3independently represent a group chosen from -OH, -OCH3, -SH, -SCH3, -OR 4 , -NH2, -NHR 5 , -NR 7 R 8 , -SO2-NH2, - SO2-NH-R 6 , SO3H, or a halogen atom.

[0114] According to a particular embodiment, R 2 and / or R 3 represent R groups 2 ' and R 3 ' corresponding to ad hoc protection groups. When R 2 ' and / or R 3 ' represent -NO2, R 2 ' and / or R 3 ' are then converted to R 2 and / or R 3 = -NH2, in particular by one of the techniques well known to those skilled in the art, for example by the action of zinc in contact with acetic acid.

[0115] Vehicle solution

[0116] This text also describes a pharmaceutically acceptable carrier solution.

[0117] This vehicle solution also makes it possible to improve the solubility of Retro-2.2 compounds as well as its analogues for subcutaneous or oral administration in order to promote their passage into the plasma of mammals. In particular, the present text describes a pharmaceutically acceptable vehicle comprising compounds of formula (I).

[0118] Indeed, the inventors have found that most of the vehicle solutions existing on the market do not satisfactorily overcome the solubility problems of Retro-2.2 compounds and its analogues. In addition, some vehicles do not allow the entire charge of molecules to be retained, which leads to leakage and then undesirable precipitation of these compounds during administration. A vehicle solution as described allows solubilization and / or maintenance in fine suspension and therefore effective administration of these compounds.

[0119] It is shown in the examples that good solubility of Retro-2.2 compounds, and its analogues, and in particular of compounds of formula (I), is obtained when these compounds are present in a pharmaceutically acceptable vehicle comprising a combination of ethyl esters of fatty acids of poppyseed oil, with castor oil. In certain embodiments, the pharmaceutically acceptable vehicle comprises (i) from 20% to 40% v / v of ethyl esters of fatty acids of poppyseed oil and (ii) from 60% to 80% v / v of castor oil.

[0120] In certain preferred embodiments, this pharmaceutically acceptable carrier comprises about 30% ethyl esters of poppyseed oil fatty acids, and about 70% castor oil.

[0121] It is also shown in the examples that good solubility of Retro-2.2 compounds, and its analogues, and in particular of compounds of formula (I), is obtained when these compounds are present in a pharmaceutically acceptable vehicle comprising a combination of dimethyl sulfoxide (DMSO), ethyl esters of fatty acids of poppyseed oil with castor oil.

[0122] Thus, according to one embodiment, the pharmaceutically acceptable carrier comprises a combination of dimethyl sulfoxide (DMSO), ethyl esters of safflower oil fatty acids with castor oil.

[0123] In some embodiments, the pharmaceutically acceptable carrier comprises (i) from 1% to 5% v / v dimethyl sulfoxide, (ii) from 20% to 40% v / v poppyseed oil fatty acid ethyl esters, and (iii) from 60% to 75% v / v castor oil.

[0124] In certain particular embodiments, this pharmaceutically acceptable carrier comprises about 3% dimethyl sulfoxide, about 29% ethyl esters of poppyseed oil fatty acids, and about 68% castor oil.

[0125] According to a particular embodiment, the ethyl esters of fatty acids in poppyseed oil are iodized. An example of iodized ethyl esters of fatty acids in poppyseed oil is Lipiodol ®, marketed by the company Guerbet. In this embodiment, the iodized ethyl esters of fatty acids are mainly in the form of ethyl monoiodo stearate and ethyl diiodostearate.

[0126] In a particular embodiment, the ethyl esters of fatty acids of poppyseed oil are a mixture of ethyl esters of fatty acids of poppyseed oil, wherein the fatty acids are selected from palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and mixtures thereof. A mixture of ethyl esters of fatty acids of poppyseed oil suitable in a carrier solution as described may thus comprise a palmitic acid ethyl ester, a stearic acid ethyl ester, an oleic acid ethyl ester, a linoleic acid ethyl ester, a linolenic acid ethyl ester, or a mixture thereof.

[0127] In a particular embodiment, the mixture of fatty acid ethyl esters of poppyseed oil comprises from 5% to 15% of palmitic acid ethyl esters, from 1% to 5% of stearic acid ethyl esters, from 8% to 15% of oleic acid ethyl esters, from 65% to 75% of linoleic acid ethyl esters, and from 3% to 7% of linolenic acid ethyl esters.

[0128] In a preferred embodiment, the mixture of fatty acid ethyl esters of poppyseed oil comprises about 10% palmitic acid ethyl esters, about 2% stearic acid ethyl esters, about 11% oleic acid ethyl esters, about 72% linoleic acid ethyl esters, and about 5% linolenic acid ethyl esters.

[0129] The inventors surprisingly discovered that the administration of Retro-2.2 compounds and its analogues in a vehicle solution of this type, in particular orally or subcutaneously, made it possible to improve the passage into the circulation and the concentration of the compounds in the plasma, and therefore their bioavailability to individuals, compared to the vehicles already known in the art.

[0130] Furthermore, the inventors discovered that the administration of compounds of formula (I) was also optimized when the latter were included in a vehicle solution as described above.

[0131] It is also shown in the examples that good solubility of Retro-2.2 compounds, and its analogues, and in particular of compounds of formula (I), is obtained when these compounds are present in a pharmaceutically acceptable vehicle comprising a combination of dimethyl sulfoxide (DMSO), with glycerol triacetate and castor oil.

[0132] Thus, in one embodiment, the pharmaceutically acceptable carrier comprises a combination of dimethyl sulfoxide (DMSO), ethyl esters of poppyseed oil fatty acids with castor oil. In some embodiments, the pharmaceutically acceptable carrier comprises

[0133] (i) 1% to 5% DMSO, (ii) 5% to 15% glycerol triacetate and (iii) 80% to 94% v / v castor oil.

[0134] In certain particular embodiments, this pharmaceutically acceptable carrier comprises about 3% DMSO, about 11% glycerol triacetate, and about 86% castor oil.

[0135] Vehicle solutions as described are suitable for administration by airborne, oral, parenteral, local, intramuscular or subcutaneous routes. In particular, the vehicle solutions are suitable for intramuscular or subcutaneous administration.

[0136] As indicated previously, the present invention also relates to a pharmaceutical composition or medicament comprising at least one compound of general formula (I) as defined previously as active ingredient, and a pharmaceutically acceptable vehicle, said pharmaceutical composition or said medicament being in particular suitable for administration by the air, oral, parenteral, local, intramuscular or subcutaneous routes.

[0137] According to a particular embodiment, the pharmaceutically acceptable vehicle is as described above.

[0138] Thus, according to a particular embodiment, the pharmaceutical composition or the medicament comprises, as a pharmaceutically acceptable vehicle, (i) from 20% to 40% v / v of ethyl esters of fatty acids of poppyseed oil and (ii) from 60% to 80% v / v of castor oil.

[0139] According to another embodiment, the pharmaceutical composition or medicament comprises, as a pharmaceutically acceptable vehicle, (i) from 1% to 5% v / v of dimethyl sulfoxide, (ii) from 20% to 40% v / v of ethyl esters of fatty acids of poppyseed oil and (iii) from 60% to 75% v / v of castor oil.

[0140] According to another embodiment, the pharmaceutical composition or medicament comprises, as a pharmaceutically acceptable vehicle, (i) from 1% to 5% of DMSO,

[0141] (ii) 5% to 15% glycerol triacetate and (iii) 80% to 94% v / v castor oil.

[0142] According to a particular embodiment, the composition or medicament comprises at least one compound of formula (I), in particular compound 1, and a pharmaceutically acceptable vehicle comprising (i) from 1% to 5% of DMSO, (ii) from 5% to 15% of glycerol triacetate and (iii) from 80% to 94% v / v of castor oil.

[0143] In particular, the composition or medicament may comprise at least the (S)-enantiomer of compound 1 ((S)-compound 1) and a pharmaceutically acceptable carrier comprising (i) from 1% to 5% DMSO, (ii) from 5% to 15% glycerol triacetate and (iii) from 80% to 94% v / v castor oil.]

[0144] In particular, the composition or medicament may comprise at least the (S)-enantiomer of compound 1 ((S)-compound 1) and a pharmaceutically acceptable carrier comprising (i) about 3% DMSO, (ii) about 11% glycerol triacetate and (iii) about 86% v / v castor oil.

[0145] According to a particular embodiment, the pharmaceutical composition or the drug may be in the form of a hydrogel.

[0146] In particular, the pharmaceutical composition or medicament in the form of a hydrogel may comprise at least one compound of formula (I) and an aqueous solution of PLGA-PEG-PLGA.

[0147] The PLGA-PEG-PLGA polymer has been described to form fluid solutions when dissolved in water at low temperatures. PLGA-PEG-PLGA solutions are capable of dissolving hydrophobic compounds. However, upon heating to 37°C, the micelles formed by the PLGA-PEG-PLGA chains interact with each other, leading to the formation of a hydrogel. This phenomenon can be exploited to generate a fluid formulation of a hydrophobic drug candidate at room temperature that will spontaneously form a hydrogel upon warming to body temperature (i.e., upon subcutaneous injection).

[0148] A hydrogel galenic comprising a PLGA-PEG-PLGA polymer of this type has been described in Vinck et al. (In Vivo Sustained Release of the Retrograde Transport Inhibitor Retro- 2.1 Formulated in a Thermosensitive Hydrogel. Int. J. Mol. Sci. 2022, 23, 14611. https: / / doi.org / 10.3390 / ijms232314611) as allowing administration of the compound Retro-2.1 in mice. This thermosensitive polymer notably allowed a prolonged release of the compound and a better control of its metabolism. The pharmaceutical composition or the medicament as described are suitable for administration by the airborne, oral, parenteral, local, intramuscular or subcutaneous routes. In particular, the pharmaceutical composition or the medicament are suitable for intramuscular or subcutaneous administration.

[0149] Definitions

[0150] As used herein, the term "about" refers to a range of values ​​within ± 10% of a specific value. For example, the term "about 120 mg" includes values ​​of 120 mg ± 10%, or values ​​from 108 mg to 132 mg.

[0151] For the purposes of this description, percentages refer to percentages by weight relative to the total weight of the formulation, unless otherwise indicated.

[0152] As used herein, the value ranges of "xy" or "x to y" include the bounds x and y and the integers between those bounds. For example, "1-5" or "1 to 5" refers to the integers 1, 2, 3, 4, and 5. Preferred embodiments include each individual integer in the value range, as well as any subcombination of those integers. For example, preferred values ​​for "1-5" may include the integers 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, etc.

[0153] As used herein, the term "pharmaceutically acceptable salt" refers to salts which are, within the scope of sound medical judgment, suitable for contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problematic complications in proportion to a reasonable benefit / risk ratio.

[0154] By pharmaceutically acceptable salt of the compounds of general formula (I) is meant in particular hydrochlorides, hydrobromides, sulfates or bisulfates, phosphates or hydrogen phosphates, acetates, oxalates, benzoates, succinates, fumarates, maleates, lactates, citrates, tartrates, gluconates, methanesulfonates, benzenesulfonates and paratoluenesulfonates.

[0155] Halogen atoms are chemical elements in group VII of the periodic table, including fluorine, chlorine, bromine and iodine.

[0156] The term alkyl radical of 1 to 3 or 4 carbon atoms denotes a linear or branched hydrogen-carbon radical; examples include methyl, ethyl, propyl, isopropyl or tert-butyl. By "PEG" or "poly(ethylene glycol)" or "polyethylene glycol" are meant in particular -(CH2-CH2-O)m- groups, optionally terminated by a group chosen from H, a C1 to C3 alkyl, or -(-O-CH2-CH2)m- groups, optionally terminated by a group chosen from H, -OH, and a C1 to C3 O-alkyl, m being chosen from 1 to 500, in particular from 4 to 500, in particular from 30 to 60.

[0157] The average molar mass of PEG may in particular be indicated after the term "PEG" after the name, for example PEG- 1450 (1450 g- mol' 1 ).

[0158] By "PLA" or "poly(lactic acid)" or "polylactic acid" is meant in particular -(C(=O)-CH(CH3)-O)p- groups, optionally terminated by a group chosen from H, a C1 to C3 alkyl, or -(O-CH(CH3)-C(=O))p- groups, optionally terminated by a group chosen from -OH, and a C1 to C3 O-alkyl, p being chosen from 1 to 2000, in particular from 5 to 500, particularly from 10 to 50.

[0159] The average molar mass of PLA can notably be indicated after the term "PLA" after the name, for example PLA- 1036 (1036 g.mol' 1 ).

[0160] By "PLGA" or "PLG" is meant a poly(lactic acid-co-glycolic acid), and in particular groups -((C(=O)-CH(CH3)-O)p-(C(=O)-CH2-O)q)r-, optionally terminated by a group chosen from H, a C1 to C3 alkyl, or groups -((O-CH2- C(=O))q-(O-CH(CH3)-C(=O))p)r-, optionally terminated by a group chosen from - OH, and a C1 to C3 O-alkyl, p, q and r being such that the PLGA polymer is random or block-type, in particular random, and in particular such that the average molar mass of the PLGA, for example indicated after the term "PLGA" after the name, is from 200 to 4000, in particular from 800 to 1200 g.mol' 1 . p, q and r are notably such that the molar percentage of lactic acid in PLGA is approximately 20%. In particular, p, q and r are such that PLGA consists of 12 units of lactic acid and 3 units of glycolic acid.

[0161] Example

[0162] I. Synthesis of compounds according to the invention

[0163] All chemicals and solvents used in the syntheses were reagent grade and were used without further purification. CH2Cl2 was distilled over calcium hydride before use. Glassware was flame-dried under vacuum and cooled under nitrogen to room temperature. All reactions were carried out under dry nitrogen and monitored by TLC. Purification was performed on a CombiFlash with a UV-vis detector and RediSep columns. Samples were adsorbed onto Celite or silica and loaded into solid-load cartridges. An ethyl acetate / cyclohexane or methanol / dichloromethane gradient was used. Fractions were collected based on detection at 254 nm.

[0164] HPLC-MS analysis and purification were performed using a Waters system (2525 Binary Gradient Module, Online Degasser, 2767 Sample Manager, 2996 Photodiode Array Detector) with a binary system for the solvent gradient. 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 loaded onto a 100-4.6mm (5mm) Zorbax SB-C18 column equilibrated with H2O / MeCN or H2O / MeOH 95:5. This system was, for example, coupled to a Waters Micromass ZQ system with a ZQ2000 quadrupole analyzer. Ionization was performed by electrospray and the other parameters were: source temperature 120 °C, cone voltage 20 V, and continuous sample injection at a flow rate of 0.3 mL per min.Mass spectra were recorded in positive and negative ion mode in the m / z 100-2000 range and processed with Mass Lynx 4.0 software.

[0165] The infrared spectra were recorded on a Spectrum Two equipped with a U ATR Two (Perkin Elmer), Diamond / ZnSe (1 reflection).

[0166] NMR analyses were performed on a Bruker Avance 400 Ultrashield spectrometer. 1H-NMR and 13C spectra were recorded at room temperature at 400 MHz and 100 MHz respectively, samples were dissolved in DMSO-d6 or CDCL at a concentration of approximately 5 mM. The DMSO singlet signal was set at 2.50 ppm. Chemical shifts are given in ppm and coupling constants in Hz. Spectral data are consistent with the associated structures.

[0167] Preparation of Compound 1: Synthesis of 3-(2,6-dihydroxyphenyl)-6-fluoro-l-methyl-2-(5- (2-methylthiazol-4-yl)thiophen-2-yl)-2,3-dihvdroquinazolin-4(lH)-one

[0168] [Table 1]

[0169] N-Methyl-5-fluoro isatoic anhydride (195 g / mol, 0.195 g, 1 mmol, 1 equiv.) obtained as described in Gupta et al. (ACS Med. Chem. Lett. 2014, 5, 1, 94-97), 2-amino-1,3-benzenediol (125 g / mol, 0.187 g, 1.5 mmol, 1.5 equiv.), and commercially available 5-(2-methyl-1,3-thiazol-4-yl)-2-thiophenecarbaldehyde (209 g / mol, 0.209 g, 1 mmol, 1 equiv.) were dissolved in acetic acid (2 mL). The mixture was heated in a microwave synthesizer at 120°C for 3 h. The crude mixture was diluted in ethyl acetate. The organic phase was washed with saturated aqueous NaHCOa solution and distilled water, then dried over magnesium sulfate, filtered, and the solvent was evaporated in vacuo.Purification on a silica gel chromatography column using an elution mixture of cyclohexane / ethyl acetate (90 / 10 to 70 / 30) allowed the recovery of ZI compound 4 from the scheme above in the form of a yellowish powder (467.5 g / mol, 0.467 g, quantitative). This compound corresponds to compound 1 according to the invention.

[0170] 1H NMR (DMSO-d6): 2.61 (s, 3H); 2.83 (s, 3H); 5.96 (s, 1H); 6.21 (dd, 1H, J = 1.1, J = 8.2); 6.42 (dd, 1H, J = 1.1, J = 8.1); 6.79 (dd, 1H, J = 4.2, J = 9), 6.86 (d, 1H, J = 3.7); 6.9 (t, 1H, J = 8.2); 7.21 (d, 1H, J = 3.6); 7.33 (td, 1H, J = 3.2, J = 8.8); 7.51 (dd, 1H, J = 3.1, J = 8.8); 7.66 (s, 1H); 9.03 (s, 1H); 9.82 (bs, 1H).

[0171] UPLC / MS: Retention time: 2.95 min,

[0172] M+ H= 468.9

[0173] Preparation of compound 2: synthesis of 3-(2,6-diaminophenyl)-6-fluoro-l-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-2,3-dihvdroquinazolin-4(lH)-one

[0174] Acetic acid, microwave

[0175] 12O c C

[0176] Compound 2 according to the invention can be obtained in a similar manner by following a synthesis protocol as previously described for compound 1 by following the synthesis scheme above.

[0177] II. Measurement of the protective activity of the compounds of the invention against different toxins and viruses

[0178] 1. Against Shiga toxin, ricin and abrin

[0179] Protocol and calculation of ECso

[0180] The compounds were tested either on A549 cells (human lung epithelial cells) or on HeLa cells (human uterine cancer cells), against Shiga toxins (Stx-1 and / or Stx-2), against ricin, or against abrin. The human cells were cultured at 37 °C in an atmosphere containing 5% CO2 in 150 cm culture flasks. 2in DMEM (Dulbecco's Modified Eagle Medium) containing 100 U / mL penicillin and 100 μg / mL streptomycin. The cells are seeded at a density of 50,000 cells per well in Cytostar-T solid scintillator bottom 96-well plates. The cells (100 μL in complete DMEM: DMEM + 10% fetal calf serum, FCS) are pre-incubated or not with the inhibitors (50 μL; different concentrations, 3 h pre-incubation). Compound 1, according to the invention, was prepared in accordance with the protocol presented previously. Compounds A to J, outside the invention, were prepared in accordance with the protocols presented in international applications WO2014 / 060586 A1 or WO2020 / 109510 A1. The structure of these compounds is indicated in the following table:

[0181] [Table 2]

[0182] These compounds, in powder form, were then solubilized in pure DMSO at a concentration of 10 mM. This solution is then diluted in the culture medium and used in the examples below in a range of concentrations. Complete medium supplemented with Shiga toxin, ricin, or abrin (50 pL, variable concentration range) is then added to each well. After a 20 h incubation, the medium (200 pL) is removed and replaced with a leucine-free DMEM medium (Eurobio) containing 10% FCS and 0.5 pCi / mL of [ 14 C]-leucine (GE). After a 7-h incubation at 37°C, the incorporation of radioactivity by the cells is determined by reading the plates using a Wallac 1450 Microbeta trilux scintillation counter (PE).

[0183] Because these toxins block protein synthesis, affected cells are no longer able to incorporate radiolabeled leucine into their proteins. In contrast, cells treated with inhibitors still synthesize proteins and therefore incorporate the radiolabeled amino acid. Because the cells concentrate the radioelement sufficiently close to the bottom of the well, this causes excitation of the scintillator contained in the plates and leads to the emission of photons detected by the scintillation counter (measured in counts per minute, cpm). These data are then expressed as the percentage of protein synthesis by the cells. Cytotoxicity curves can thus be plotted without an inhibitor or in the presence of an inhibitor.Data analysis by nonlinear regression allows the ICso to be estimated in the absence or presence of the compound, i.e. the concentration of toxin for which 50% assimilation of radioactive leucine is observed, which corresponds to 50% of viable cells. The higher the ICso value, the greater the cellular protection, since a higher concentration of toxin is required to generate the same cytotoxicity.

[0184] The curve of ICso values ​​as a function of compound concentrations allows the calculation of ECso, which represents the concentration of product giving 50% of its maximum antitoxin protective effect. The lower the ECso, the more effective the compound.

[0185] 2. With regard to poxyirus vaccinia and Myxomatosis

[0186] Cytotoxicity of compounds

[0187] RK13 (ATCC CCL-37) (for myxomatosis virus) and HeLa (ATCC-CCL2) (for vaccinia virus) cells were cultured in DMEM without phenol red (DI 145; Sigma Aldrich) supplemented with 10% SVE (Eurobio-Scientific), 1 mM sodium pyruvate (S8636; Sigma Aldrich), L-Glutamine (G7513; Sigma Aldrich) and Penicillin-Streptomycin solution (P0781; Sigma Aldrich).

[0188] Cells were seeded at 6000 to 8000 cells per well in a Corning Cellbind 96-well plate in complete DMEM medium. 24 h after seeding, cells were treated with compounds 1 and A to J for a series of concentrations. Cells were then incubated at 37 °C and 5% CO2 for 3 to 6 days.

[0189] Post-treatment, cells were labeled with Image-iT DEAD Green Viability Stain (Invitrogen 110291) and MitoTracker Orange (Invitrogen M7510) for 30 minutes at 37°C. Cells were fixed with 4% formalin (Sigma) for 10 min, washed with PBS, and incubated with PBS Hoechst 33342 (1 mg / mL).

[0190] High-content microscopy data acquisition is performed on a Thermo CellInsight CX7 HCS microscope, using a compartmental analysis algorithm. Results are extracted, normalized to untreated conditions, and expressed as the average of three independent wells + / - SD.

[0191] The 50% cytotoxic concentration (CC50), which corresponds to the concentration of the administered compound that reduces cell viability by 50%, was determined for each compound. The lower the CC50 value, the more toxic the compound is to cells.

[0192] Antiviral activity

[0193] Antiviral activity was measured using ANCHOR technology (NeoVirTech). Cells were cultured under the same conditions as for the cytotoxicity assay, treated at 8 concentrations in triplicate and infected with the myxomatosis-derived virus MYXV-T1-ANCHOR (MOI 0.5) and the vaccinia-derived virus VacV-ANCHOR (MOI 0.1).

[0194] Three to four days post-infection the cells are fixed under the same conditions as previously described, and incubated with PBS Hoechst 33342 (1 mg / mL).

[0195] Plaques are imaged using a Thermo Scientific CellInsight CX7 HCS microscope. Compartmental analysis coupled with a Spot Detector algorithm is used to detect and quantify the infection rate (number of fluorescent cells over total number of cells) and the replication rate (intensity of ANCHOR spots).

[0196] Results are obtained after measurement and automated analysis of a minimum of 2000 cells per well per replicate. Results are extracted, normalized to infected / untreated conditions and expressed as the average of three independent wells + / - SD. In the same manner as in point ILL, the ECso was determined for each compound.

[0197] 3. Results

[0198] The ECso results for each of the compounds against the indicated toxins and viruses are given in the following table.

[0199] [Table 3]

[0200] These results show that the compounds of formula (I), and in particular compound 1 according to the invention, have very low EC50s. They exhibit improved properties with regard to cellular protection against Shiga toxins, ricin and abrin and against vaccinia and myxomatosis viruses, in particular compared to equivalent compounds of the prior art.

[0201] It is further demonstrated that the compounds of formula (I), and in particular compound 1 according to the invention, have high CC50 values, synonymous with low cytotoxicity of these compounds towards the treated cells.

[0202] Finally, the calculated solubility expressed in log S was estimated by in silico calculation for each of the compounds. The higher the value of log S, the greater the solubility of the molecule. These results show that the compounds according to the invention, in particular compound 1, have improved solubility.

[0203] In conclusion, these results show that compounds according to the invention, such as compound 1, exhibit both effective inhibitory activity against the various Shiga toxins Stx-1, Stx-2, ricin and abrin, as well as against the various vaccinia pox and myxomatosis viruses, without posing any cytotoxicity problem for the treated cells. In addition, these characteristics, in particular solubility, are improved with the compounds according to the invention compared to those obtained for compounds of the prior art.

[0204] III. Measurement of the pharmacokinetic activity of Retro-2.2 analogues and compounds of formula (I) in a first vehicle solution (DMSO / sunflower oil fatty acid ethyl esters / castor oil)

[0205] The pharmacokinetic activity (plasma concentration of the molecule (nM) versus time (h)) of the following analogues of the Retro-2.2 molecule was tested: compound 1 (white circles), Retro-2.2 (compound B from the table above) (white squares), compound C (white triangles) from the table above and compound E (white diamonds), also recalled in the table above. The compounds were administered to female BALB / c mice orally (gavage) (Figure 1) and subcutaneously (Figure 2) at a concentration of 33 mg / kg in a Lipiodol © / castor oil mixture in mass proportions of 30% / 70%. The plasma concentration of these compounds was measured by liquid chromatography with tandem mass spectrometry (LC-MS-MS).The results of these tests show that compound 1 according to the invention has the best pharmacokinetic properties among all the molecules tested since it allows the best exposure in terms of concentration of the molecule, and this for a prolonged duration. In addition, it is observed that the relative bioavailability of compound 1 according to the invention is better when administered subcutaneously, since the area under the curve is greater compared to oral administration.

[0206] Furthermore, the pharmacokinetic activity of the Retro-2.2 compound was compared to that of compound 1 according to the invention following subcutaneous administration in female BALB / C mice at a concentration of 33 mg / kg in a vehicle solution comprising a Lipiodol © / castor oil mixture in mass proportions of 30% / 70% (formulation B) or in a vehicle solution as described in the text comprising a DMSO / Lipiodol © / castor oil mixture in mass proportions of 3% / 29% / 68%

[0207] (formulation C).

[0208] To prepare formulations C, the Retro-2.2 molecules and compound 1 according to the invention, in powder form, were solubilized in pure DMSO. The solution was homogenized by vortexing for 30 seconds at maximum speed. Lipiodol © was then added. Again, the solution was homogenized by vortexing for 30 seconds at maximum speed. Castor oil was added gently with a pipette. The solution was homogenized by inverting the tube containing it 20 times. It was then sonicated in a water bath at 37 °C for 30 minutes. Again, the solution was homogenized by inverting the tube containing it 20 times.

[0209] The results are reported in Figure 3 according to the following representation: Retro-2.2 in formulation B (white squares), Retro-2.2 in formulation C (black squares), compound 1 according to the invention in formulation B (white circles) and compound 1 according to the invention in formulation C (black circles).

[0210] The results of these tests show that for both the Retro-2.2 molecule and compound 1 according to the invention, the relative bioavailability is improved when the molecule is administered in formulation C. Furthermore, these results show that compound 1 according to the invention has an even better relative bioavailability than that of the Retro-2.2 molecule, regardless of the vehicle solution used.

[0211] Finally, a simulation of a repeated administration of the Retro-2.2 molecule and compound 1 according to the invention was produced on the basis of the results obtained from the pharmacokinetic parameters of the single administration tests. These tests were carried out by subcutaneous administration in female BALB / c mice at a concentration of 33 mg / kg, twice a day in a formulation C comprising a DMSO / Lipiodol © / castor oil mixture in mass proportions 3% / 29% / 68%.

[0212] The pharmacokinetic parameters obtained with Retro-2.2 or compound 1 according to the invention in formulation C administered subcutaneously were used to simulate the plasma concentrations that would be obtained following two subcutaneous injections per day, 6 hours apart (Figure 4).

[0213] The results show that the ECso of Retro-2.2 against the toxin ricin, abrin or Stx is approximately 20 or 30 nM. For a compound 1 according to the invention, the ECso is around or below 20 nM. Therefore, the simulation suggests that twice-daily subcutaneous injections of Retro-2.2 or a composition according to the invention should achieve concentrations well ten times higher than their respective ECso against these toxins for a good part of the day. This criterion must be met to validate a molecule in early drug development studies.

[0214] The same is true for their activity against pox viruses.

[0215] VI. Measurement of the pharmacokinetic activity of Retro-2.2 analogues and compounds of formula (I) in a second vehicle solution (DMSO / glycerol triacetate / castor oil)

[0216] The pharmacokinetic activity (concentration of the molecule in plasma (pM) as a function of time (h)) of the (S)-enantiomer of compound 1 according to the invention was tested. The compound was administered to female BALB / c mice subcutaneously at a concentration of 33 mg / kg in a mixture of DMSO 3% / Glycerol triacetate 11% / Castor oil 86%. The plasma concentration of the compound was measured by liquid chromatography with tandem mass spectrometry (LC-MS-MS). The calculated pharmacokinetic parameters are provided in the table below [Table 4]

[0217] The results of these tests show that (S)-compound 1 ((S)-enantiomer of compound 1) has particularly satisfactory pharmacokinetic properties over a prolonged period.

[0218] Then, compound 1 and other analogues of the Retro-2.2 molecule mentioned above (compound A, compound B, compound C and compound E) were incubated with liver microsomes of female CD1 mice in the presence or absence of NADPH, the cofactor of CYP450s, at a final concentration of 5 pM for 45 minutes at 37 °C and 300 rpm (Thermomixer). Samples were collected at different times and added to cold acetonitrile to stop the reaction and precipitate proteins. The samples were centrifuged, the supernatant diluted and analyzed by LC-MS-MS in the presence of a known concentration of Retro-2.1 as an internal standard.

[0219] As shown in Figure 5, compound 1 exhibits slower metabolism compared to other compounds, particularly in the presence of mouse liver microsomes. The compounds are stable in the absence of NADPH (results not shown).

[0220] From the experimental curves, the inventors then proceeded to simulate repeated injections of the (S)-enantiomer of compound 1 at a dose of 33 mg / kg once or twice a day. These simulations made it possible to achieve a theoretical plasma concentration of 500 times and 1000 times the ECso of (S)-compound 1 in vitro (Figure 6).

[0221] Subsequently, subcutaneous injection of the (S)-enantiomer of compound 1 at a dose of 33 mg / kg in Balb / ca mice resulted in a compound concentration in organs ranging from 67 to 350 times the ECso against Shiga toxins of (S)-compound 1 in vitro. At doses of 1 to 3 mg / kg, once or twice daily, plasma and lung concentrations reached 75 to 175 nM, i.e., 12 to 29 times the ECso of (S)-compound 1 in vitro. They were 50 to 125 nM in the lungs after 2 h, i.e., 8 to 21 times the ECso of (S)-compound 1 in vitro.

[0222] Compound 1 was identified in vivo in different organs of mice treated subcutaneously at 33 mg / kg as previously described, namely in the brain, lungs, liver, small intestine, colon, kidneys and plasma. Two hours after administration of compound 1, the concentrations of compound 1 measured by LC-MS-MS were as follows:

[0223] Brain: 1.7 pM

[0224] Lungs: 0.5 pM

[0225] Liver: 0.6 pM

[0226] Small intestine: 2.1 pM

[0227] Colon: 0.8 pM

[0228] Kidneys: 0.4 pM

[0229] Plasma: 0.7 pM

[0230] These results show that the administration of compound 1 by subcutaneous route, in particular in the formulation DMSO 3% / Glycerol triacetate 11% / Castor oil 86% makes it possible to obtain a concentration theoretically protective of mice against pox viruses or Shiga toxins.

[0231] V. Metabolite of a compound of formula (I)

[0232] The structure of the major metabolite of compound 1 identified in the culture supernatants after incubation in the presence of liver microsomes (see above) was determined by LC-MS-MS and then synthesized for confirmation according to methods known in the art by those skilled in the art, for example according to a procedure analogous to the synthesis method described in Forrester et al. (Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2. Nat Chem Biol. 2020 Mar;16(3):327-336. doi: 10.1038 / s41589-020-0474-4. Epub 2020 Feb 17. PMID: 32080624; PMCID: PMC7039708).

[0233] The compound obtained is as follows:

[0234] This compound, also called a metabolite, was also identified in vivo in the kidney, liver, colon, lungs and brain of female BALB / c mice after subcutaneous treatment with compound 1 at a concentration of 33 mg / kg in a DMSO / safflower oil fatty acid ethyl ester / castor oil solution as described in Example III. Two hours after treatment, the following concentrations of compound 1 were measured by LC-MS-MS in the different organs:

[0235] Kidney: 0.5pM

[0236] Eoie: IpM

[0237] Colon: <200 nM

[0238] Lungs: <200 nM

[0239] Brain: 1.5 pM

Claims

Demands 1. Compound with general formula (I): Or - p is equal to 1, 2 or 3; - R 1 represents at each occurrence, independently, a hydrogen atom, a halogen atom, an alkoxy radical of 1 to 3 carbon atoms, including a methoxy group, -NO2, or -NH2; - R 2 and R 3 independently represent a group chosen from -OH, -OCH3, -SH, -SCH3, -OR 4 , -NH2, -NHR 5 -NR 7 R 8 , -SO2-NH2, -SO2-NH-R 6 , SO3H, or a halogen atom; - R 4 , R 5 and R 6 independently represent a group of formula (II) - L-(X)i-(PEG)-(Y)jZ, where: - i and j independently represent 0 or 1; - L represents -C(=O)- or -C(=O)-(CH2)kC(=O)-, with k being equal to 1, 2 or 3, in particular 2; - R 7and R 8 represent, independently of each other, a linear or branched Ci to C3 alkyl; - X and Y independently represent either a poly(lactic acid) or a poly(lactic acid-co-glycolic acid); - PEG represents poly(ethylene glycol); - Z represents a group chosen from H, a C3-Ci alkyl, -OH, a C3-Ci O-alkyl, or -L-Re, where L is defined as before and Re is a residue of formula (I) related to the said -L-(X)i-(PEG)-(Y)j- group defined beforehand via its R group 2 or R 3 , said group R 2 or R 3 being -OH, -NH2 or -SO2-NH2; - R 9 represents -CH3 or -CH2-OH, in particular represents CH3. as well as stereoisomeric forms, mixtures of stereoisomeric forms or their pharmaceutically acceptable salts.

2. Compound of general formula (I) according to claim 1, wherein R9 represents -CH3.

3. General formula compound (I) according to any one of claims 1 or 2, in which p is equal to 1.

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

5. General formula compound (I) according to any one of the preceding claims wherein R 2 and R 3 independently represent a group chosen from -OH, -OCH3, -SH, -SCH3, -OR 4 , -NH2, -NHR 5 -NR 7 R 8 , -SO2-NH2, -SO2-NH- R 6 , SO3H, in particular R 3 , represents an -OH group, where R 4 , R 5 , R 6 , R 7 and R 8 are as defined in claim 1.

6. Compound of general formula (I) according to any one of the preceding claims, of the following general formula (la): where R 2 is such as defined in claim 1.

7. Compound of general formula (I) according to any one of claims 1 to 5, of the following general formula (Ib): in which R 2 and R 3 are chosen from the following combinations: R 2 is a -OH group and R 3 is a group -NH2, R 2 is a -OH group and R 3 is a group -SO2NH2, R 2 is a -OH group and R 3 is a group -SH, R 2 is a -OH group and R 3 is a group -SO3H, R 2 is a -OH group and R 3 is a group -SMe, R 2 is a -OH group and R 3 is a group - OME, R 2 is a -OH group and R 3is a halogen atom, and in particular is a fluorine atom, R 2 is a group -NH2 and R 3 is a group -NH2, R 2 is a group - NH2 and R 3 is a group -SO2NH2, R 2 is a group -NH2 and R 3 is a group -SH, R 2 is a group -NH2 and R 3 is a group -SO3H, R 2 is a group - NH2 and R 3 is a group -SMe, R 2 is a group - NH2 and R 3 is a group -OMe, R 2 is a group - NH2 and R 3 is a halogen atom, and in particular is a fluorine atom, R 2 is a group -SH and R 3 is a group -SH, R 2 is a group -SH and R 3 is a group -SO2NH2, R 2 is a group -SH and R 3 is a group -SO3H, R 2 is a group -SH and R 3 is a group -SMe, R 2 is a group -SH and R 3 is a group - OME, R 2 is a group -SH and R3 is a halogen atom and in particular is a fluorine atom, R 2 is a group -OMe and R 3 is a group -OMe, R 2 is a group -OMe and R 3 is a group -SMe, R 2 is a group -OMe and R 3 is a halogen atom, and in particular is a fluorine atom, R 2 is a group -SMe and R 3 is a group -SMe, R 2 is a group -SMe and R 3 is a halogen atom, and in particular is a fluorine atom, and R 2 is a halogen atom and in particular is a fluorine atom and R 3 is a halogen atom and in particular is a fluorine atom, where Me is a methyl.

8. General formula compound (I) according to any one of the preceding claims, of the following formula:

9. Compound of general formula (I) according to any one of claims 1 to 8, for its use in the prevention and / or treatment of disorders induced by toxins with an intracellular mode of action using retrograde transport, or by viruses or bacteria using retrograde transport and / or dependent on syntaxin 5 to infect cells, in particular viruses or bacteria entering cells by endocytosis, or by intracellular parasites.

10. A compound for use according to claim 9, characterized in that said toxins with an intracellular mode of action using retrograde transport are selected from the ricin, abrin, Shiga toxin and Shiga-like toxins produced by Shigella dysenteriae and E. coli, pertussis toxin, subtle cytotoxin and thermolabile enterotoxin.

11. Compound for its use according to claim 9, characterized in that said viruses are pox viruses, in particular smallpox virus, monkeypox virus, vaccinia virus and leporipoxviruses, in particular myxomatosis virus, adeno-associated viruses, in particular serotype 2, polyomaviruses, in particular JC polyomavirus and BK polyomavirus, papillomaviruses, filoviruses, in particular Ebola virus and Marburg virus, enteroviruses, in particular enterovirus 71, herpesviruses, in particular Herpes simplex virus type 2 and cytomegalovirus (hCMV), viruses of the genus Arenavirus, in particular lymphocytic choriomeningitis virus, and pneumoviruses, in particular respiratory syncytial virus.

12. Pharmaceutical composition or medicinal product comprising at least one compound of general formula (I) as defined in any one of claims 1 to 8 as an active ingredient, and a pharmaceutically acceptable vehicle, said pharmaceutical composition or medicinal product being in particular suitable for administration by the air, oral, parenteral, local, intramuscular or subcutaneous routes.

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