Compound ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro- 2H-pyrrolo[3,4-b]quinoline-3-carboxylate, salt, crystalline form, cocrystal, formulation, processes for preparation, application as medicaments, pharmaceutical compositions and new use particularly as inhibitor of aurora kinases.

The invention thus relates to the compound ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate, salt, crystalline form, cocrystal, formulation, processes for preparation thereof, application thereof as medicaments, pharmaceutical compositions and new uses thereof particularly as Aurora kinase inhibitors.

The compound ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate in free base form, referred to hereinafter as mpound F, conforms to the formula (F) below:

The preparation of this compound (F) is described hereinafter as intermediate E1 in example 1 of the present invention described below.

The present invention accordingly provides the compound ethyl 8-oxo-9-[3-(1 H- benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrol o[3,4-b]quinoline-3- carboxylate or compound F of formula (F) below:

this compound F being in the form:

- of sulphate salt compound I of said compound F,

- or of cocrystal compound II of said product of formula (F)

- or of formulation comprising said compound of formula (F) or said sulphate salt compound I of the product of formula (F) or said cocrystal compound II of the product of formula (F), as defined above.

The present invention accordingly provides the compound I as defined above or sulphate salt of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate, which therefore has the formula (I) below:

The present invention accordingly provides the compound I as defined above or sulphate salt of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate, characterized in that it is in crystalline form.

The present invention accordingly provides the compound II as defined above or cocrystal of the product of formula (F), ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate.

The present invention accordingly provides the compound II as defined above or cocrystal of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro- 2H-pyrrolo[3,4-b]quinoline-3-carboxylate, characterized in that it is the cocrystal with L- malic acid.

The present invention likewise provides the formulations as defined above, characterized in that they comprise said compound of formula (F), ethyl 8-oxo-9-[3-(1 H- benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrol o[3,4-b]quinoline-3- carboxylate, or said sulphate salt compound I of the product of formula (F) or said cocrystal compound II of the product of formula (F), as defined above.

The compounds I and II or formulations as defined above that are provided by the invention are selective inhibitors of Aurora kinases.

The present invention likewise relates to pharmaceutical compositions comprising these compounds.

These compounds may be used as anti-cancer agents.

The invention also relates to processes for obtaining these compounds and to some of the intermediates in said processes.

[Technical problem]

A number of cancer treatment strategies are aimed at inhibiting the Aurora-type kinases, particularly Aurora A and B, which are involved in the regulation of mitosis; in this regard, see Nature Reviews 2004, 4, 927-936; Cancer Res. 2002, 94, 1320; Oncogene 2002, 21 , 6175; Mol. Cell. Biol. 2009, 29(4), 1059-1071 ; Expert Opin. Ther. Patents 2005, 15(9), 1 169-1 182; Clin. Cancer Res. 2008, 14(6), 1639).

A number of Aurora inhibitor compounds (for example, MLN-8237 from Millennium, AZD-1 152 from Astra-Zeneca or SNS-314 from Sunesis) are presently under evaluation in clinical trials. MLN-8237 is selective for Aurora A, while AZD-1 152 is selective for Aurora B. Since both kinases, Aurora A and B, are deregulated in cancer, inhibiting both Aurora A and B provides an advantage over selective inhibition of one kinase or the other. Moreover, multi-kinase compounds are in existence, such as the compound AT-9283 from Astex, which inhibit a number of kinases, including Aurora A and B. For this type of compound it is difficult to predict that the inhibition of the Aurora kinases could actually be exploited clinically, because the inhibition of kinases other than Aurora A and B is likely to give rise to side-effects. One technical problem the invention intends to solve, therefore, is that of developing a compound which is a potent and selective inhibitor of Aurora A and B.

The cyclic nucleotide phosphodiesterase enzyme PDE3 plays a major part in the signalling mediated by the cyclic nucleotides cAMP and cGMP that takes place in the myocytes of the smooth cardiac and vascular muscles. The inhibition of PDE3 by small molecules has an inotropic and vasodilatory action, which may prove to be useful on a short-term basis for the treatment of certain cardiomyopathies in which defects in cardiac contraction are a feature. It has been shown, however, that the long-term use of these molecules increases mortality in this type of patient. Furthermore, the use of PDE3 inhibitors in patients who do not have this type of pathology, such as patients affected by cancer, may give rise to unwanted effects on cardiac rhythm. It is therefore important, in the context of an anti-cancer therapy, not to inhibit PDE3. In this regard, see Exp. Opin. Invest, drugs 2002, 1 1 , 1529-1536 "Inhibitors of PDE3 as adjunct therapy for dilated cardiomyopathy"; Eur. Heart J. supplements 2002, 4(supplement D), D43-D49 "What is wrong with positive inotropic drugs? Lessons from basic science and clinical trials". Another technical problem the invention intends to solve is that the Aurora A and B inhibitor compound is not to inhibit the enzyme PDE3.

It is important as well that the anti-cancer agent presents a metabolic stability (see section 10.2.2 of "Chimie pharmaceutique" [Pharmaceutical chemistry], G.L.Patrick, De Boeck, published 2003, ISBN 2-7445-0154-9). The reason is that the inadequacy of the pharmacokinetics of pharmaceutical compounds is one of the primary reasons for failure in their development (Curr. Pharm. 2005,1 1 , 3545 "Why drugs fail— a study on side effects in new chemical entities"). Moreover, the metabolism is often a major determinant of clearance, of drug interactions, of intra-individual variability in pharmacokinetics, and of clinical efficacy and toxicity (Curr. Drug Metab. 2004, 5(5), 443-462 "Human hepatocytes in primary culture: the choice to investigate drug metabolism in man"). Another technical problem the invention intends to solve is that the Aurora A and B inhibitor compound is to exhibit high chemical and metabolic stability.

[Prior art]

Bioorg. Med. Chem. Lett. 2002, 12, 1481 -1484 describes in table II the compound 6A, which has a different tricyclic structure.

WO 01/36422 describes compounds having a different tricyclic structure.

WO 2004/005323 describe formula (a):

as an EPO receptor having a different tricyclic structure. Furthermore, the compound does not include a phenyl ring substituted by the group -O-benzimidazolyl at the top of the trycyclic ring system.

WO 2005/016245 describes anti-cancer compounds having a different tricyclic structure, of formula (b):

in which R4 can represent a substituted phenyl group. Substitution by the group -O-benzimidazolyl is neither described nor suggested.

WO 2007/012972 and EP 1746097 describe anti-cancer compounds of formula

(c)

(c)

and, in accordance with one embodiment, according to the formula (c'):

(C)

R2 represents a substituted aryl or heteroaryl group, X represents N or CR7, R5 and R6 may both represent H or CH ₃ . No example in WO 2007/012972 contains the

group which characterizes the compound of formula (I). Moreover, among the compounds resolved, WO 2007/012972 teaches that it is the dextrogyratory compounds which are the most active on Aurora A or B (cf. examples 1 19 and 120 in the table on page 147).

WO 02/062795 describes co (d):

(d)

in which R4 and R5 may optionally form a 5- or 6-membered ring. [Brief description of the invention]

The crystalline sulphate salt and the cocrystal that are subject-matter of the present invention have demonstrated a greatly enhanced (solubility, stability) physicochemical profile, which has allowed the development of formulations compatible with administration to human beings. The crystalline sulphate salt or cocrystal thus formulated has demonstrated a significant activity on xenografted models of human tumours in mice.

The advantages provided by the present invention are in particular:

the identification of a sulphate salt, crystalline salt or compound I, of a compound II and of formulations, as defined above, that are subject- matter of the present invention, as selective inhibitors of Aurora kinase proteins;

this sulphate salt or compound I is crystalline whereas the base and many other salts such as, for example, hydrochloride, phosphate, p- toluenesulphonate and naphthalene-1 ,5-disulphonate of this product are amorphous;

this salt or compound I, the compound II and formulations, that are subject-matter of the present invention, are much more soluble in aqueous medium than the amorphous base or compound F, and therefore allow better formulation properties for administration to human beings.

The advantages of the sulphate salt and of the cocrystal with L-malic acid are therefore, in particular, those of being more stable than the base under storage conditions at high temperature and humidity. Crystalline compounds are therefore easier to weigh and distribute than the amorphous base. The advantages of the formulation are in particular those of compatibility with intravenous (iv) administration to human beings: the formulation allows a reduction in the incidence of an oxidation impurity (example 3) and the provision of a solution which is stable at 5°C for several months and is compatible with clinical use.

The present invention provides in particular the formulations as defined above, characterized in that they are in the form of a micellar solution.

The present invention particularly provides the formulations as defined above, characterized in that they are in the form of a micellar solution compatible with intravenous administration.

Analytical methods used for the present invention

Method LC/MS-A The products for analysis are separated on an Acquity Beh C18 UPLC column, 1.7 μιτι, 2.1x50 mm (Waters) thermostated at 70°C and eluted at a flow rate of 1 ml/min with a gradient from acetonitrile containing 0.1 % formic acid (solvent B) into water containing 0.1 % formic acid (solvent A); elution program: isocratic stage at 5% of solvent B for 0.15 min, gradient from 5 to 100% of solvent B in 3.15 min, then return to the initial conditions over 0.1 min. The products are detected by an Acquity PDA diode-array UV/vis detector (Waters, wavelength range scanned: 192-400 nm), a Sedex 85 light scattering detector (Sedere, nebulizing gas: nitrogen, nebulizing temperature: 32°C, nebulizing pressure 3.8 bar) and an Acquity SQD mass spectrometer (Waters, operating in positive and negative mode, mass range scanned: 80 to 800 amu).

Method LC/MS-B

The spectra were obtained on a Waters UPLC-SQD apparatus in positive and/or negative electrospray ionization mode (ES+/-), under the following liquid chromatography conditions: column: ACQUITY BEH C18 1.7 μιτι, 2.1x50 mm; T _C0 | _Umn : 50°C; flow rate: 1 ml/min: solvents: A: H?Q (0.1 % formic acid) B: CH^CN (0.1 % formic acid): gradient (2 min): 5 to 50% B in 0.8 min; 1 .2 min: 100% of B; 1.85 min 100% of B; 1.95 min 5% of B.

¹ H NMR

The spectra are recorded on a Bruker spectrometer, the product being dissolved in DMSO-d6. The chemical shifts δ are expressed in ppm.

IR

The infrared spectrum is recorded at between 4000 and 400 cm ^"1 on a Nicolet Nexus spectrometer as a KBr disk, with a resolution of 2 cm ^"1 .

Measurement of the optical rotation

The optical rotations were recorded on a Perkin-Elmer 341 polarimeter.

Elemental analysis

The elemental analyses were conducted on a Thermo EA1 108 analyser.

Powder X-ray diffraction

The analyses were carried out on a Bruker D8-Advance diffractometer in Bragg- Brentano configuration. The data were recorded at 21 °C (ambient temperature) using a CuKal (λ=1.5406 A) X-ray source generated at 35kV - 40mA, variable-divergence slits regulated to irradiate a sample surface with a width of 4 mm, an Anton-Paar TTK450 temperature chamber and a Vantec PSD detector. The sample is placed in a gate of the Anton Paar TTK450 temperature chamber, on a silicon slide. The sample is flattened by rotating movements on the silicon slide in order to give a planar surface of constant thickness. The diffractograms were recorded in the 2Θ angle range of 2°-40° in continuous mode, using 2Θ angular increments of 0.033° and an acquisition time per step of 0.5 s.

HPLC method for the studies of chemical stabilities and solubilities

The products under analysis are separated on an XTerra MS C18 HPLC column, 3.5 μιη 4.6x50 mm (Waters) thermostated at 40°C and eluted at a flow rate of 1.2 ml/min with a gradient from acetonitrile containing 0.035% trifluoroacetic acid (solvent A) into water containing 0.05% trifluoroacetic acid (solvent B) with the following elution program: gradient from 10 to 95% of solvent A in 25 min then isocratic stage for 5 min, then return to the initial conditions in 2 min, and column re-equilibration stage lasting 5 min. The products are detected using an Agilent diode-array UV/VIS detector (wavelength range scanned: 190-950 nm),

Measurement of solubility

The solubility of the compounds is measured in ultra-purified water, in an HCI-buffered medium of pH 1 , in an acetate-buffered medium of pH 4.7 and in a phosphate-buffered medium of pH 7.4.

The ultra-purified water is obtained from a Millipore production system.

The HCI-buffered medium of pH 1 is prepared by diluting to 1/10 a ready-to-use HCI 1 N solution sold by Merck.

The acetate-buffered medium of pH 4.7 is prepared by adding 0.75 g of sodium acetate trihydrate to 0.35 ml of pure acetic acid, which is made up to 250 ml with ultra-purified water.

The phosphate-buffered medium of pH 7.4 is prepared by adding 40.5 ml of 0.1 M disodium hydrogenphosphate solution to 9.5 ml of 0.1 M sodium dihydrogen phosphate solution, which is then made up to 100 ml with ultra-purified water.

Whichever medium in which the solubility of the compound is measured, the protocol employed was as follows: Approximately 4 mg of compound in powder form are weighed out into a Perkin Elmer 7 ml glass tube. A volume of 2 ml of the medium in question is added. Stirring is then applied to the tube for 16 hours at ambient temperature in the absence of light, using a Rock'n'roller RM5 stirrer. After 16 hours of stirring, the suspensions are transferred to 2 ml safe lock Eppendorf tubes and centrifuged for 15 min at 18000 rpm in a Sigma 3-18k centrifuge. An aliquot of the supernatant is taken immediately after centrifuging, for measurement of the concentration of compound by HPLC, using the experimental conditions described above.

Study of physicochemical stability of the solid form

The chemical and physical stability of the solid form of the compound in question is studied by HPLC and by powder X-ray diffraction after 14 days of storage of the compound under four stress conditions: 50°C/ambient humidity, 50°C/80% relative humidity, 80°C/ambient humidity and 80°C/80% relative humidity.

Approximately 20 mg of compound in question for the study are placed in four crystallizers. Two crystallizers are closed non-hermetically and placed in two ovens (brand name, model) temperature-controlled at 50°C and 80°C respectively. Two crystallizers are left open and placed in two desiccators containing a pre-saturated KCI solution which generates a relative humidity in the desiccator of -80%. The two desiccators are then placed in two Heraeus UT6060 ovens temperature-controlled at 50°C and 80°C respectively.

After 14 days of storage, the crystallizers are closed hermetically and their temperature is brought naturally to 21 °C (ambient temperature). The four samples of the compound in question are then analysed by powder X-ray diffraction to verify the physical stability, and by HPLC to verify the chemical stability, of the compound.

Biological methods

Measurement of the activity on Aurora A and B

The capacity to inhibit the kinase activity of the enzyme is estimated by measuring the residual kinase activity of the enzyme in the presence of different concentrations of the test compound (generally 0.17 to 10000 nM). A dose-response curve is produced, which allows an IC ₅ o (50% inhibitory concentration) to be ascertained. The kinase activity is measured by a radioactive assay of the amount of radioactive phosphate (33P) incorporated into a fragment of the protein NuMA (Nuclear Mitotic Apparatus protein) after 30 min of incubation at 37°C. The test compound is first dissolved at different concentrations in dimethyl sulphoxide (DMSO). Reaction takes place in the wells of a FlashPlate microtitre plate (Nickel Chelate FlashPlate-96, PerkinElmer). Each well (100 μΙ) contains 10 nM Aurora A, 500 nM NuMA, 1 μΜ ATP and 0.2 μθί ΑΤΡ-γ-33Ρ in a buffer of 50 mM Tris-HCI, pH=7.5; 10 mM MgCI ₂ ; 50 mM NaCI, 1 mM dithiothreitol. The final percentage of DMSO is 3%. After homogenization by stirring, the plate is incubated at 37°C for 30 minutes. The contents of the wells are then removed and the wells are washed with PBS buffer. The radioactivity is then measured using a TRILUX I450 Microbeta counter (Wallac). In each plate, there are eight control wells: four positive controls (maximum kinase activity), for which measurement is made in the presence of enzyme and substrate and in the absence of compound of the invention, and four negative controls (background noise), for which measurement is made in the absence of enzyme, substrate and test compound. The measurements are given in Table I.

Aurora A

The recombinant human enzyme Aurora A used is expressed in entire form with a poly-Histidine tag in N-terminal position and is produced in E.coli. A fragment (amino acids 1701-21 15) of the human protein NuMA, with a poly-Histidine tag in C-terminal position, is expressed in recombinant form in E.coli.

Aurora B/lncenp

The entire human enzyme Aurora B is co-expressed with a fragment of the human protein Incenp (aa 821-918) in a baculovirus system and is expressed in insect cells. Aurora B has a poly-Histidine tag in N-terminal position, while the Incenp fragment possesses a Glutathione-S-Transferase (GST) tag in N-terminal position. The two proteins form a complex which is called Aurora B/lncenp. A fragment (aa1701-21 15) of the human protein NuMA with a poly-Histidine tag in C-terminal position is expressed in recombinant form in E.coli. This fragment is used as substrate.

The present invention further provides any process for preparing the compounds I, II and formulation as defined above.

The present invention accordingly provides any process for preparing the compound I as defined above or sulphate salt of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te, and particularly the process as defined in scheme 1 above or else the processes as described in the experimental section.

The present invention accordingly provides any process for preparing the compound II as defined above or cocrystal of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te, and particularly the processes as described in the experimental section.

The present invention accordingly provides any process for preparing a formulation as defined above, and particularly the processes as described in the experimental section.

Experimental section according to the present invention

Example 1 : preparation of the sulphate of ethyl 8-oxo-9-r3-(1 H-benzimidazol-2- yloxy)phenvn-4,5,6,7,8,9-hexahvdro-2H-pyrrolor3,4-b1quinolin e-3-carboxylate, laevogyratory enantiomer

Scheme 1

Intermediate A1 : 2-Chloro-1-(tetrahvdropyran-2-yl)-1 H-benzimidazole

A 10 I reactor is charged under argon and with stirring with 2.5 I of THF, 180 g of 2- chlorobenzimidazole (1.18 mol) and 325 ml of 3,4-dihydro-2H-pyran (6.56 mol, 3 eq.). The reactor is heated until dissolution takes place (temperature of the medium: 40°C). Then 6.3 g of para-toluenesulphonic acid (0.033 mol, 0.028 eq.) are introduced. The temperature is maintained at between 49 and 52°C for 2.5 hours. The system is cooled to 12°C and 7.65 g of sodium methoxide (0.142 mol, 0.12 eq.) are added, with stirring maintained, over a total time of 15 minutes. The temperature is then brought to 18°C, 5 I of n-heptane are added, and the whole is filtered over 300 g of Clarcel FLO-M, the retentate being washed with 5 I of n- heptane. The filtrate is concentrated to dryness under reduced pressure to give 292.6 g of 2- chloro-1-(tetrahydropyran-2-yl)-1 H-benzimidazole in the form of a slightly yellow oil (quantitative yield). ¹ H NMR (400 MHz, DMSO-d6): 1.42 to 2.01 (m, 5H); 2.21 to 2.34 (m, 1 H); 3.69 to 3.78 (m, 1 H); 4.12 (d, J=1 1.4 Hz, 1 H); 5.72 (dd, J=2.4 and 1 1.2 Hz, 1 H); 7.22 to 7.34 (m, 2H); 7.62 (d, J=7.2 Hz, 1 H); 7.78 (d, J=7.2 Hz, 1 H).

Intermediate B1 : 3-ri-(Tetrahvdropyran-2-yl)-1 H-benzimidazol-2-yloxy1- benzaldehyde

Two 2 I three-necked round-bottomed flasks, each equipped with a condenser, a thermometer and a stirring shaft, are charged under argon with N,N-dimethylformamide (0.4 I per flask), and with 3-hydroxybenzaldehyde (68.5 g, flask 1 ; 64.2 g, flask 2; 1.08 mol). Sodium hydride (60% dispersion in mineral oil) is then added in portions (flask 1 : 26 g; flask 2: 24 g; 1.25 mol, 1.2 eq.), the maximum temperature during the addition being 32°C. 2-Chloro-1- (tetrahydropyran-2-yl)-1 H-benzimidazole (A1 ), with an estimated purity of 85%) is then introduced (flask 1 : 151 g in 0.5 I of N,N-dimethylformamide; flask 2: 142 g in 0.5 I of N,N- dimethylformamide; 1.05 mol, 0.97 eq.). The mixture is then taken to reflux (temperature 140°C, temperature rise time 40 min) and the reflux is maintained for 1 hour. Heating is then stopped and the mixture is left to cool for 1.5 hours. The contents of the two flasks are combined. The whole is mixed slowly with 5 I of iced-water. The aqueous phase obtained is subsequently extracted with 4 x 2.5 I of ethyl acetate (AcOEt). The organic phases are then combined, washed with 3 I of water and then with 2 I of saturated NaCI solution, and finally dried by addition of MgS0 ₄ overnight. The organic phase obtained is subsequently filtered on a glass frit (porosity 4) and concentrated to dryness under reduced pressure, to give 385 g of a brown oil (LC/MS-A, tr (retention time)=1.86 min, MS positive mode: m/z=323.16).

A fraction of 158 g of the crude material obtained above is dissolved hot in 1.5 I of an n-heptane/AcOEt mixture (8/2 by volume), combined with 500 g of silica (70-30 mesh) and the whole is stirred for 45 minutes. The suspension obtained is filtered on Celite and washed with 3 I of an n-heptane/AcOEt mixture (8/2 by volume). The organic phase obtained is concentrated to dryness under reduced pressure. The residue is resuspended in 200 ml of isopropyl ether by mechanical stirring and treatment with ultrasound, and then filtered on a glass frit (porosity 3). The solid obtained is washed with 2x40 ml of ispropyl ether and dried under reduced pressure at 40°C for 16 hours, to give 68 g of solid. A similar treatment applied to the rest of the crude product produces 87.8 g of solid. The solids obtained are combined and homogenized to give 155.8 g of 3-[1-(tetrahydropyran-2-yl)-1 H-benzimidazol-2- yloxy]benzaldehyde in the form of light-beige crystals (LC/MS-A, tr=1.87 min, MS positive mode m/z=323.13). MS(LC/MS-B): tr=1 .00 min; [M+H]+: m/z 323; ¹ H NMR (400 MHz, DMSO- d6): 1.54 to 1 .62 (m, 1 H); 1 .63 to 1 .84 (m, 2H); 1.92 to 2.03 (m, 2H); 2.30 to 2.42 (m, 1 H); 3.70 to 3.79 (m, 1 H); 4.10 (d, J=1 1.5 Hz, 1 H); 5.74 (dd, J=2.1 and 1 1.1 Hz, 1 H); 7.13 to 7.22 (m, 2H); 7.43 (d, J=7.3 Hz, 1 H); 7.65 (d, J=7.3 Hz, 1 H); 7.73 (t, J=7.8 Hz, 1 H); 7.78 to 7.83 (m, 1 H); 7.87 (d, J=7.8 Hz, 1 H); 7.96 (s, 1 H); 10.05 (s, 1 H).

Washing the silica phases used above with 2 I of an n-heptane/AcOEt mixture (1/1 by volume) produces 67 g of material after concentration to dryness under reduced pressure. This material is taken up in 2 I of an n-heptane/AcOEt mixture (9/1 by volume), combined with 285 g silica (70-30 mesh), stirred and treated with ultrasound for 1 hour. The suspension is then filtered on Celite, and the solid phase is washed with 2 I of an n-heptane/AcOEt mixture (9/1 by volume). The filtrate is concentrated to dryness under reduced pressure and the residue is triturated in 400 ml of an n-heptane/ethanol mixture (95/5 by volume), filtered on a glass frit (porosity 3) and dried under reduced pressure, to give 35 g of 3-[1-(tetrahydropyran- 2-yl)-1 H-benzimidazol-2-yloxy]benzaldehyde in the form of light-beige crystals. (LC/MS-A, tr=1.93 min, MS positive mode m/z=323.16). Intermediate C1 : ethyl 8-Oxo-9-{3-ri-(tetrahvdropyran-2-yl)-1 H-benzimidazol-2- yloxylphenyl}-4,5,6,7,8,9-hexahvdro-2H-pyrrolor3,4-b1quinoli ne-3-carboxylate

A 2 I Erlenmeyer flask is charged, with magnetic stirring, with 50 g of 3-amino-2- ethoxycarbonylpyrrole hydrochloride and 0.204 I of 2N sodium hydroxide. The whole is stirred at ambient temperature (AT) for 15 minutes and then extracted with 3 x 0.3 I of dichloromethane. The organic phases are combined, dried over MgS0 ₄ and concentrated to dryness under reduced pressure. The residue is triturated with n-pentane, filtered and dried under reduced pressure to constant weight, to give 36.4 g of 3-amino-2-ethoxycarbonylpyrrole in the form of a brown solid.

A 2 I three-necked round-bottomed flask equipped with a stirring shaft, a thermometer and a condenser is charged with 1.2 I of 1-butanol, 145 g of 3-[1-(tetrahydropyran-2-yl)-1 H- benzimidazol-2-yloxy]benzaldehyde (0.405 mol, B1 ), 62.4 g of 3-amino-2- ethoxycarbonylpyrrole (1 eq., 0.405 mol), 46.8 g of 97% 1 ,3-cyclohexanedione (1 eq., 0.405 mol) and 70.5 ml of N,N-diisopropylethylamine (1 eq.) and this initial charge is taken to reflux (temperature rise time 55 minutes, reflux held for 30 minutes, temperature 1 14°C). The mixture is subsequently cooled to AT and concentrated to dryness under reduced pressure, to give 290 g of a brown oil containing ethyl 8-oxo-9-{3-[1-(tetrahydropyran-2-yl)-1 H- benzimidazol-2-yloxy]phenyl}-4,5,6,7,8,9-hexahydro-2H-pyrrol o[3,4-b]quinoline-3-carboxylate. (LC/MS-A, tr=1 .96 min, MS positive mode m/z=553.33). A similar operation carried out with 35 g of 3-[1-(tetrahydropyran-2-yl)-1 H-benzimidazol-2-yloxy]benzaldehyde (0.098 mol, example B1 ) gives 72 g of a brown oil containing ethyl 8-oxo-9-{3-[1-(tetrahydropyran-2-yl)- 1 H-benzimidazol-2-yloxy]phenyl}-4,5,6J,8,9-hexahydro-2H-pyrro lo[3,4-b]quinoline-3- carboxylate. (LC/MS-A, tr=1 .96 min, MS positive mode m/z=553.35).

Intermediate D1 : Ethyl 8-oxo-9-r3-(1 H-benzimidazol-2-yloxy)phenvn-4,5,6,7,8,9-

A 2 I round-bottomed flask is charged with 224 g of the brown oil containing ethyl 8- oxo-9-{3-[1-(tetrahydropyran-2-yl)-1 H-benzimidazol-2-yloxy]phenyl}-4,5,6,7,8,9-hexahydro- 2H-pyrrolo[3,4-b]quinoline-3-carboxylate (example 1 .3), 0.7 I of ethanol and 0.243 I of 2N hydrochloric acid. The mixture is stirred at AT for 16 hours, then filtered on a glass frit (porosity 4). The filtrate is concentrated to dryness under reduced pressure and the residue is triturated with 0.5 I of isopropyl ether. The solid obtained is dried under reduced pressure at a constant weight, to give 253 g of a brown solid containing ethyl 8-oxo-9-[3-(1 H-benzimidazol- 2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quino line-3-carboxylate. (LC/MS-A, tr=1 .46 min, MS positive mode m/z=469.29). A similar operation carried out with 54 g of the brown oil containing ethyl 8-oxo-9-{3-[1-(tetrahydropyran-2-yl)-1 H-benzimidazol-2- yloxy]phenyl}-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate (C1 ) produces 60 g of a brown solid containing ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te. (LC/MS-A, tr=1.48 min, MS positive mode m/z=469.29).

An aliquot fraction of 0.8 g of the material obtained may be purified by chromatography on a cartridge of 50 g of silica (10-90 μιη) (Biotage SNAP, KP-Sil) eluted with an isocratic stage of dichloromethane for 20 minutes, then a gradient from 0 to 1 % by volume of isopropanol in dichloromethane over one hour, and finally an isocractic stage of dichloromethane/isopropanol (99/1 by volume) for 20 minutes. The fractions containing the expected product are combined, to give 0.21 g of a yellow solid. The products of two similar chromatographic separations carried out on the same scale are recrystallized from acetonitrile to give a total of 0.16 g of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4, 5,6, 7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate in the form of beige crystals. (LC/MS-A, tr=1 .61 min, MS positive mode m/z=469.28). ¹ H NMR (400 MHz, DMSO-d6): 1.29 (t, J=7.0 Hz, 3H); 1.80 to 1 .97 (m, 2H); 2.19 to 2.27 (m, 2H); 2.55 to 2.69 (m, 1 H); 2.81 (dt, J=4.8 and 17.2 Hz, 1 H); 4.26 (q, J=7.0 Hz, 2H); 5.1 1 (s, 1 H); 6.73 (d, J=3.3 Hz, 1 H); 7.02 to 7.16 (m, 5H); 7.25 (t, J=7.9 Hz, 1 H); 7.31 to 7.38 (m, 2H); 8.34 (s, 1 H); 1 1.33 (broad s, 1 H); 12.26 (broad s, 1 H). Elemental analysis: C= 68.72%; H= 5.10%; N= 1 1.82%; H ₂ 0= 0.38%.

Intermediate E1 : Laevogyratory enantiomer of ethyl 8-οχο-9-Γ3-(1 Η- benzimidazol-2-yloxy)phenvn-4,5,6,7,8,9-hexahvdro-2H-pyrrolo r3,4-b1 uinoline-3-

The laevogyratory enantiomer is purified from the crude product of example D1 on a Welk-01 RR chiral column, 10 μΜ, 80x350 mm (Regis, USA) eluted with an n- heptane/dichloro-methane/ethanol/triethylamine mixture (50/47.5/2.5/0.1 by volume). The elution of the products is detected by UV spectroscopy at 265 nm. Quantities of 10 g of the crude product described in example D1 are injected in each operation. Under these conditions, the peak corresponding to the laevogyratory enantiomer is eluted with a tr of between 50 and 80 min. The fractions of purified laevogyratory enantiomer corresponding to the operations needed to purify 310 g of the crude product described in example D1 are combined, homogenized and concentrated to dryness under reduced pressure, to yield 50 g of a beige solid. Mass spectrum (LC/MS-B): tr=0.77 min; [M+H]+: m/z 469; [M-H]-: m/z 467. ¹ H NMR (400 MHz, DMSO-d6): 1 .29 (t, J=7.1 Hz, 3H); 1.79 to 1 .97 (m, 2H); 2.19 to 2.27 (m, 2H); 2.55 to 2.66 (m, 1 H); 2.81 (dt, J=4.9 and 17.1 Hz, 1 H); 4.26 (q, J=7.1 Hz, 2H); 5.12 (s, 1 H); 6.73 (d, J=3.4 Hz, 1 H); 7.02 to 7.16 (m, 5H); 7.25 (t, J=8.3 Hz, 1 H); 7.29 to 7.41 (m, 2H); 8.32 (s, 1 H); 1 1 .31 (broad s, 1 H); 12.26 (broad s, 1 H). IR: principal bands: 1678; 1578; 1525; 1442; 1 188; 1043 and 743 cm ^"1 . Optical rotation: [a] _D = -38.6+0.7 at C=0.698 mg/ml in methanol. Elemental analysis: C= 68.18%; H= 5.92%; N= 1 1.22%; H ₂ 0= 1.25%. Compound I: sulphate of the laevogyratory enantiomer of ethyl 8-οχο-9-Γ3-(1 Η- benzimidazol-2-yloxy)phenvn-4,5,6,7,8,9-hexahvdro-2H-pyrrolo r3,4-b1guinoline-3- carboxylate

In a round-bottomed flask, 350 ml of acetonitrile are added to 3.9 g (8.33 mmol) of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate in amorphous base form, and the whole is stirred at 60°C for 30 minutes, and 20 ml of acetonitrile are added while the mixture is kept with stirring at 60°C. A slightly hazy, brown solution is obtained. This solution is then filtered on a glass frit of porosity 3. Then 85 ml of 0.1 M sulphuric acid in a water/acetonitrile mixture (1/4-v/v; 8.5 mmol, 1.02 eq.) are added and the mixture is briefly stirred by hand and is allowed to return to ambient temperature (~25°C, over 30 minutes) without stirring. The crystals formed are filtered and washed with 3x10 ml of acetonitrile and then dried under reduced pressure at 70°C to constant weight, to give 3.1 g of yellow crystals, melting point: 242-244°C, monosulphate form determined by microanalysis, containing 5.7% H ₂ 0 (1 .8 mol/mol, determined by the K. Fischer method) and 0.5 mol/mol of acetonitrile (determined by NMR).

The crystals are then combined with 70 ml of ethyl acetate and the mixture is taken to reflux for 30 minutes. After return to ambient temperature, the crystals are collected by filtration, washed with 30 ml of ethyl acetate and dried under reduced pressure at 70°C overnight. This gives 3.04 g of yellow crystals: melting point 242-244°C, acetonitrile: 0.2 mol/mol (NMR), H ₂ 0 5.9% (1 .9 mol/mol).

The crystals are then combined again with 120 ml of ethyl acetate and the mixture is taken to reflux for 1 h 30 min. After return to ambient temperature, the crystals are collected by filtration, washed with 30 ml of ethyl capacitate and dried under reduced pressure at 70°C overnight. This gives 2.94 g of yellow crystals: monosulphate of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate.

Melting point 242-244°C

1H NMR spectrum (400 MHz, δ in ppm, DMSO-d6):

1.29 (t, J=7.1 Hz, 3 H); 1 .78 to 1 .96 (m, 2 H); 2.20 to 2.28 (m, 2 H); 2.61 (m, 1 H); 2.81 (m, 1 H); 4.26 (q, J=7.1 Hz, 2 H); 5.12 (s, 1 H); 6.73 (d, J=3.4 Hz, 1 H); 7.04 to 7, 18 (m, 5 H); 7.27 (t, J=8.0 Hz, 1 H); 7.34 to 7.42 (m, 2 H); 8.33 (s, 1 H); 1 1.31 (broad s, 1 H). Acetonitrile: traces

Infrared spectrum:

The principal bands are centred at: 3424; 3209; 2939; 2621 ; 1684; 1635; 1578; 1471 ; 1372; 1 188; 1042; 782; 762; 586 cm ^"1

Elemental analysis:

C = 54.41 - 54.43%; H = 5.06 - 4.94%; N = 9.51 - 9.53%; S = 5.24 - 5.25%; water = 5.47% (K. Fischer)

Optical rotation:

OR = -10.1 +/- 0.6; C = 2.204 mg / 0.5 ml CH ₃ OH at 589 nm; temperature 20°C

The present invention accordingly provides the compound I as defined above, or sulphate salt of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate, characterized in that its melting point is 242-244°C,

Example 2. a: preparation of the cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate with L-malic acid (compound II).

The cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate with L-malic acid (compound II) is obtained by grinding 0.53 millimol of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate (intermediate E1 from example 1 ) with 0.53 millimol of L-malic acid in the presence of 100 microlitres of ethanol, by means of a bead mill, at ambient temperature for 20 minutes.

Example 2.b: preparation of the cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate with L-malic acid (compound II).

The cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate with L-malic acid (compound II) is obtained by grinding 0.53 millimol of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate (intermediate E1 from example 1 ) with 0.53 millimol of L-malic acid in the presence of 100 microlitres of ethanol, by means of a bead mill, at ambient temperature for 20 minutes. The resulting solid is suspended in 2.5 ml of ethyl acetate at ambient temperature for 24 hours. Example 2.c : preparation of the cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate with L-malic acid (compound II).

5 The cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate with L-malic acid (compound II) is obtained by mixing an ethanolic solution (0.3 millimol in 250 microlitres) of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te (intermediate E1 of example 10 1 ) with an ethanolic solution (0.3 millimol in 56 microlitres) of L-malic acid. The crystallization is initiated with 2 mg of cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H- benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrol o[3,4-b]quinoline-3-carboxylate with L-malic acid, and then 250 microlitres of ethyl acetate are added. The suspension obtained is left with stirring overnight at ambient temperature.

15

Example 3: preparation of ethyl 8-oxo-9-r3-(1 H-benzimidazol-2-yloxy¾phenyll-

5,6,7,8-tetrahvdro-2H-pyrrolor3,4-b1quinoline-3-carbox late.

De

[3- -4,

[3,

0

In order to ensure a reproducible yield in the preparation described below, it is advisable to apply operating conditions which aim to prevent the presence of traces of humidity in the equipment and the reactants during the oxidation reaction. A three-necked flask equipped with a magnetic bar is charged in succession with 2.03 g of dextrogyratory 5 enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H- pyrrolo[3,4-b]quinoline-3-carboxylate, isolated by the chromatographic method described in example 1 , 100 ml of anhydrous acetonitrile from a new bottle opened at the time, and 1 g of 2,3-dichloro-5,6-dicyano. The reaction mixture is stirred at ambient temperature. The solution blackens and then a precipitate appears. After 30 minutes of reaction, the reaction mixture is 0 concentrated under reduced pressure and the residue is taken up in 100 ml of water. The resulting solution is extracted with 5x100 ml of ethyl acetate. The organic phases are combined, dried over magnesium sulphate and concentrated under reduced pressure. The residue is taken up in a minimal volume of a dichloromethane/methanol mixture (1/1 , v/v), combined with 6 g of chromatography-grade silica (40-63 μιη) and concentrated again. The resulting power is placed in a chromatography cartridge, which is subsequently installed at the top of a column of 100 g of silica (10-90 μιη, Biorad), and the assembly is fitted to a Biotage chromatography instrument. The column is eluted with a gradient from 0 to 5% of isopropanol in dichloromethane in 10 column volumes in accordance with the standard conditions given by the Biotage chromatography software. The homogeneous fractions are combined and concentrated under reduced pressure, to give 1.77 g of a yellow solid. The solid is taken up in 150 ml of saturated aqueous sodium hydrogen carbonate solution and extracted with 3 x 150 ml of ethyl acetate. The organic phases are combined, washed with 150 ml of saturated aqueous sodium hydrogen carbonate solution, dried over magnesium sulphate and concentrated under reduced pressure. The previous aqueous phases are combined, extracted with 1x150 ml of toluene, and then 2x150 ml of a toluene/ethyl acetate mixture (1/1 , v/v), and the organic phases are combined, washed with 150 ml of saturated aqueous sodium hydrogen carbonate solution, dried over magnesium sulphate and concentrated under reduced pressure. The concentrated residues of the organic phases are resuspended in 50 ml of ethyl acetate and the suspension is taken to reflux for 15 minutes and then allowed to cool to ambient temperature. The solid formed is filtered and dried under reduced pressure, to give 970 mg of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 5,6,7,8-tetrahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate. SAR216905 VAC. LDS2.207.1 , LC/MS: method LC/MS-A, tr=1.35 min, MS positive mode m/z=467.33,

1 H NMR spectrum (400 MHz, d in ppm, DMSO-d6): 1.34 (t, J=7.1 Hz, 3 H); 2,10 (m, 2 H); 2.61 (m, 2 H); 3.15 (m, 2 H); 4.32 (q, J=7.1 Hz, 2 H); 7.1 1 (m, 2 H); 7.19 (m, 1 H); 7.35 (m, 1 H); 7.38 to 7.44 (m, 4 H); 7.50 (t, J=7.9 Hz, 1 H); 12.78 (extended m, 2 H)

Biological results:

Example 4: demonstration of the crystalline structure of compounds I and II

The crystalline or amorphous nature of compound I (sulphate), of compound II (co- crystal) and of the intermediate E1 is studied by powder X-ray diffraction.

The diffractogram recorded for the intermediate E1 is characterized by a diffusion halo throughout the angular range studied (2° - 40° to 2Θ) which is characteristic of an amorphous product.

The diffractogram recorded for compound I, as obtained in accordance with the experimental conditions described in example 1 , is characteristic of a crystalline product. The diffractogram recorded is shown in Figure 4-1 and the details of the principal diffraction peaks are given in Table 4-1 .

The present invention accordingly provides the compound I as defined above or sulphate salt of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate having the X-ray diffractogram as defined in Figure 4-1 below.

The diffractogram recorded for compound II, as obtained in accordance with the experimental conditions described in example 2, is characteristic of a crystalline product. The diffractogram recorded is shown in Figure 4-2 and the details of the principal diffraction peaks are given in Table 4-2.

The present invention accordingly provides the compound II as defined above or cocrystal with L-malic acid of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te having the X-ray diffractogram as defined in Figure 4-2 below:

Figure 4-1 : Powder X-ray diffractogram recorded for compound I as obtained in accordance with the experimental conditions detailed in example 1.

Table 4-1 : 2Θ angular position, interplanar spacing and relative intensity of the principal diffraction peaks recorded for compound I as obtained in accordance with the experimental conditions detailed in example 1.

Figure 4-2: Powder X-ray diffractogram recorded for compound II (cocrystal) as obtained in accordance with the experimental conditions detailed in example 2.

2Θ O d (A) Intensity

4.797 18.40588 14.9

1 1.476 7.7043 33.2

1 1.848 7.46367 15.1

12.7 6.96481 13.2

14.232 6.21834 7.4

14.837 5.96602 12.8

15.825 5.59552 1 1.6

17.086 5.18542 33.2

18.589 4.76942 20

18.966 4.67532 35.9

19.444 4.56146 18.1

20.209 4.39061 100

21.094 4.20842 48.3

21.539 4.12238 29.7

22.1 19 4.01562 87.3

23.121 3.84384 19.9

25.001 3.5588 35.6

26.139 3.40638 43.6

29.326 3.0431 35.1

Table 4-2: 2Θ angular position, interplanar spacing and relative intensity of the principal diffraction peaks recorded for the compound II (cocrystal) as obtained in accordance with the experimental conditions detailed in example 2.

Example 5: Determination of the solubility of compounds I and II

The solubility of compound I and compound II is measured in ultra-purified water, in a pH 1 HCI-buffered medium, in a pH 4.7 acetate-buffered medium and in a pH 7.4 phosphate- buffered medium.

The table below collates the solubility values measured after 16h of stirring for compound I and compound II.

*: Saturation not reached

Table 5-1 : Solubility values for compounds I and II, measured after 16 h of stirring an ultra-purified water, in a pH 1 HCI buffer, in a pH 4.7 acetate buffer and in a pH 7.4 phosphate buffer (target 2 mg/ml). Example 6: determination of the stability of compounds I and II under different conditions

The physical and chemical stability of compound I and compound II is studied by powder X-ray diffraction and by HPLC respectively, after 14 days of storage under four stress conditions: 50°C/ambient humidity, 50°C/80% relative humidity, 80°C/ambient humidity and 80°C/80% relative humidity.

Figure 6-1 compares the powder X-ray diffractograms recorded for the four aliquots of compound I taken from the four crystal I izers, stored for 14 days under the four different humidity and temperature conditions. The diffractograms recorded for the aliquots kept for 14 days at 50°C/ambient humidity, 50°C/80% relative humidity and 80°C/ambient humidity are comparable with the diffractogram recorded for compound I as received. The diffractogram recorded for the aliquot kept for 14 days at 80°C/80% relative humidity is different from that recorded for the compound I as received. These results demonstrate the physical stability of compound I over 14 days under the conditions of 50°C/ambient humidity, 50°C/80% relative humidity and 80°C/ambient humidity, and the physical instability of compound I over 14 days under conditions of 80°C/80% relative humidity.

Table 6-1 reports the percentage degradation, determined by HPLC for the four aliquots of compound I stored under the four stress conditions. These results demonstrate the chemical stability of compound I over 14 days under conditions of 50°C/ambient humidity, 50°C/80% relative humidity and 80°C/ambient humidity, and the chemical instability of compound I over 14 days under conditions of 80°C/80% relative humidity.

Figure 6-1 : Powder X-ray diffractograms recorded for the aliquots of compound I stored for 14 days under conditions of 50°C/ambient humidity (HN), 50°C/80% relative humidity (HR), 80°C/ambient humidity (HN) and 80°C/80% relative humidity (HR), compared with the diffractogram of compound I recorded as received.

Table 6-1 : Percentage degradation of compound I stored for 14 days under stress conditions of 50°C/ambient humidity, 50°C/80% relative humidity, 80°C/ambient humidity and 80°C/80% relative humidity

Figure 6-2 compares the powder X-ray diffractograms recorded for the four aliquots of compound II taken from the four crystal I izers, stored for 14 days under the four different humidity and temperature conditions. The diffractograms recorded for the aliquots kept for 14 days at 50°C/ambient humidity, 50°C/80% relative humidity, 80°C/ambient humidity and 80°C/80% relative humidity are comparable with the diffractogram recorded for compound I as received. These results demonstrate the physical stability of compound II over 14 days 5 under the conditions of 50°C/ambient humidity, 50°C/80% relative humidity, 80°C/ambient humidity, and 80°C/80% relative humidity.

Table 6-2 reports the percentage degradation, determined by HPLC for the four aliquots of compound II stored under the four stress conditions. These results demonstrate the chemical stability of compound II over 14 days under conditions of 50°C/ambient humidity, 10 50°C/80% relative humidity and 80°C/ambient humidity, and the chemical instability of compound II over 14 days under conditions of 80°C/80% relative humidity.

Figure 6-2: Powder X-ray diffractograms recorded for the aliquots of compound II 15 stored for 14 days under conditions of 50°C/ambient humidity (HN), 50°C/80% relative humidity (HR), 80°C/ambient humidity (HN) and 80°C/80% relative humidity (HR), compared with the diffractogram of compound II recorded as received.

Table 6-2: Percentage degradation of compound II stored for 14 days under stress conditions of 50°C/ambient humidity, 50°C/80% relative humidity, 80°C/ambient humidity and 80°C/80% relative humidity Example 7: Formulation with 5 mg/ml (active ingredient) in micellar solution for i.v administration

A round-bottomed flask is charged with 25 mg of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate (example 1 , intermediate E1 ), 375 mg of polysorbate 80 (Tween 80), and 125 μΙ of ethanol. The suspension is stirred for around 15 minutes until dissolution takes place, and then 4.5 ml of 5% aqueous glucose solution (G5) containing 17.5 mg of ascorbic acid (for the solution with 3.5 mg/ml of ascorbic acid) or 2.5 mg of ascorbic acid (for the solution with 0.5 mg/ml of ascorbic acid) are added. The mixture is acidified to a pH of 2 using H2SO4 and then stirred until complete homogeneity is obtained. Following dissolution, the pH is raised to 3 by addition of 1 N NaOH. The volume, lastly, is made up to 5 ml by addition of G5.

The formulation is kept under nitrogen in antibiotic flasks for storage at 4°C in the absence of light.

The appearance of the oxidation product ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-5,6,7,8-tetrahydro-2H-pyrrolo[3,4-b]quinoline- 3-carboxylate (example 3) is monitored by HPLC. The results of the assay of the oxidation product (example 3) after 35 days of storage at 5°C are given in the table below, by comparison with a reference solution of the sulphate of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate (example 1 , compound I) at 5 mg/ml in a water/acetonitrile mixture (50/50).

Example 8: Formulation with 5 mg/ml (of active product) formed from the sulphate of the laevogyratory enantiomer of ethyl 8-oxo-9-r3-(1 H-benzimidazol-2- yloxy)phenvn-4,5,6,7,8,9-hexahvdro-2H-pyrrolor3,4-b1guinolin e-3-carboxylate (example 1 , compound I) in micellar solution for i.v administration

A round-bottomed flask is charged with 300 mg of ascorbic acid, then with 8 ml of 5% glucose (G5). The suspension is stirred until a solution is obtained, and then 250 μΙ of ethanol and 750 mg of polysorbate 80 or Solutol HS15 (BASF) are added. The mixture is stirred until complete homogeneity is obtained, and then 60 mg of the sulphate of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H- pyrrolo[3,4-b]quinoline-3-carboxylate (example 1 , compound I) are added. The mixture is stirred for 30 min until dissolution takes place. Then 250 μΙ of 1 N NaOH are added to adjust the pH to 3. The volume, finally, is made up to 10 ml by addition of G5.

Packaging must be done in non-actinic glass for storage. A program of stability of three months at 5°C with protection from light was performed for the Solutol formulation; no significant increase in the oxidation product ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-5,6,7,8-tetrahydro-2H-pyrrolo[3,4-b]quinoline- 3-carboxylate (example 3) was observed by HPLC assay.

The invention likewise provides a process for preparing the crystalline sulphate salt and the cocrystal and the formulation as defined above. The present invention accordingly provides a process for preparing compound I as defined in Claims 1 to 6 or sulphate salt of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate, as defined in scheme 1 below:

The present invention accordingly provides a process for preparing the compound II as defined above, characterized in that:

- alternatively the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate is ground with

L-malic acid in the presence of ethanol,

- or the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te is ground with L-malic acid in the presence of ethanol, and the resulting solid is suspended in ethyl acetate, - or an ethanolic solution of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H- benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrol o[3,4-b]quinoline-3-carboxylate is mixed with an ethanolic solution of L-malic acid, then crystallization is initiated with the cocrystal of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2- yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoli ne-3-carboxylate with L-malic acid, and then ethyl acetate is added.

The present invention accordingly provides a process for preparing a formulation as defined above, characterized in that:

- either the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te, polysorbate 80 (Tween 80) and ethanol are introduced and stirred and then an aqueous glucose solution containing ascorbic acid is added and then the pH of the mixture is adjusted to 3,

- or ascorbic acid, then 5% glucose are introduced and stirred, then ethanol and polysorbate 80 or Solutol are added, the mixture is stirred and the sulphate of the laevogyratory enantiomer of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate (compound I) is added, and the mixture is stirred and 1 N NaOH is added to adjust the pH to 3.

Preparation examples for the sulphate salt, its crystalline forms and formulations that are subject matter of the present invention are described in the present invention.

In the text below, the starting materials and the reactants, when their preparation is not described, are available commercially or described in the literature, or else may be prepared in accordance with methods which are described therein or which are known to the skilled person.

Unexpectedly, indeed, it has been shown that the sulphate salt and the cocrystal with L- malic acid of the compound of formula (F) possess stability and solubility properties which are further enhanced relative to the same compound in base form.

Lastly, the sulphate salt and the cocrystal with L-malic acid are more soluble than the compound of formula (F) in free base form, as shown by the analyses described in example 5.

It has also been shown that the sulphate form of the compound of formula (F) and the cocrystal with L-malic acid possess enhanced temperature stability properties relative to the form of other salts (hydrochloride, phosphate, p-toluenesulphonate, naphthalene- 1 ,5-disulphonate, all obtained in amorphous form) of this same compound.

From these analyses it is evident that the sulphate salt of the compound and the cocrystal with L-malic acid of formula (F) exhibit both solubility properties and stability properties that are greater than those of the compound of formula (F) in the form of the base or a hydrochloride, phosphate, p-toluenesulphonate or naphthalene-1 ,5- disulphonate salt, and which make it particularly well suited to the manufacture of medicaments.

The physicochemical properties of the compound of formula (F) in sulphate salt or L- malic acid cocrystal form also allow it to be kept under the usual conditions, without excessively onerous precautions in relation to the presence of light, temperature and humidity.

A formulation in micellar solution has been shown which is suited to the intravenous administration of the compound of formula (F), allowing the compound of formula (F) to be stabilized for a number of weeks to a number of months, and this solution is even more stable when it is produced from the sulphate form of the compound of formula (F). The sulphate salt of the compound I according to the present invention is an anti-cancer agent. The cocrystal of the compound II according to the present invention is an anticancer agent. The same applies to the formulations according to the present invention. In this regard, compound I, II and the formulations according to the present invention can be used for preparing medicaments, especially medicaments intended for treating cancers, especially those mentioned below.

Accordingly, in another of its aspects, the invention provides medicaments which comprise the sulphate salt, the cocrystal or the formulations defined above. These medicaments are used therapeutically, particularly in the treatment of cancers, especially those mentioned below.

A further subject of the invention is therefore the use of the sulphate salt for preparing a medicament intended for treating the cancers referred to below.

According to another of its aspects, the present invention relates to pharmaceutical compositions comprising the sulphate salt as active principle. These pharmaceutical compositions include an effective dose of the sulphate salt, and also at least one pharmaceutically acceptable excipient. Said excipients are selected, according to the pharmaceutical form desired and the administration route desired, from the customary excipients which are known to the skilled person.

The invention therefore extends to pharmaceutical compositions comprising as active principle at least one of the medicaments as defined above.

Such pharmaceutical compositions of the present invention may further comprise, where appropriate, active principles of other antimitotic medicaments such as, more particularly, those based on taxol, cis-platin, DNA intercalating agents and others.

These pharmaceutical compositions may be administered orally, parenterally or locally by topical application to the skin and the mucous membranes, or by intravenous or intramuscular injection.

These compositions may be solid or liquid and may take any of the pharmaceutical forms that are commonly used in human medicine, such as, for example, plain or film- coated tablets, pills, lozenges, gel capsules, drops, granules, injectable preparations, ointments, creams or gels; they are prepared according to the usual methods. The active principle may be incorporated in these preparations in excipients which are normally employed in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fats of animal or plant origin, paraffin derivatives, glycols, various wetting agents, dispersants or emulsifiers, and preservatives.

The usual dosage, which is variable according to the product used, the individual treated and the condition in question, may be, for example, from 0.05 to 5 g per day in adults, or preferably from 0.1 to 2 g per day.

The present invention accordingly provides a medicament characterized in that it comprises the compound I as defined above in the form of the sulphate salt of ethyl 8- oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate.

The present invention accordingly provides a medicament characterized in that it comprises the compound II as defined above in the form of the cocrystal of ethyl 8-oxo- 9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4- b]quinoline-3-carboxylate.

The present invention accordingly provides a medicament characterized in that it comprises a formulation of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]- 4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxyla te as defined above.

The present invention further provides a pharmaceutical composition characterized in that it comprises, as active principle, the compound I or sulphate salt of ethyl 8-oxo-9- [3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4-b]quinoline- 3-carboxylate as defined above and also at least one pharmaceutically acceptable excipient.

The present invention further provides a pharmaceutical composition characterized in that it comprises, as active principle, the compound II or cocrystal of ethyl 8-oxo-9-[3- (1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrr olo[3,4-b]quinoline-3- carboxylate as defined above and also at least one pharmaceutically acceptable excipient.

The present invention further provides a pharmaceutical composition characterized in that it comprises, as active principle, a formulation of ethyl 8-oxo-9-[3-(1 H-benzimidazol- 2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quino line-3-carboxylate as defined above and also at least one pharmaceutically acceptable excipient. The present invention additionally provides for the use of the compound I as defined above, or sulphate salt of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate for preparing a medicament intended for treating cancer.

The present invention additionally provides for the use of the compound II as defined above or cocrystal of ethyl 8-oxo-9-[3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9- hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate for preparing a medicament intended for treating cancer.

The present invention additionally provides for the use of a formulation of ethyl 8-oxo-9- [3-(1 H-benzimidazol-2-yloxy)phenyl]-4,5 _! 6 _! 7 _! 8 _! 9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3- carboxylate as defined above, for preparing a medicament intended for treating cancer.

A medicament of this kind may in particular be intended for the treatment or prevention of a disease in a mammal.

The present invention particularly provides for the use of the compounds I or II or formulation as defined above for preparing a medicament intended for the prevention or treatment of diseases associated with uncontrolled proliferation.

The present invention more particularly provides for the use of the compounds I or II or formulation as defined above for preparing a medicament intended for the treatment or prevention of a disease selected from the following group: blood vessel proliferation disorders, fibrotic disorders, mesangial cell proliferation disorders, metabolic disorders, allergies, asthmas, thromboses, nervous system diseases, retinopathy, psoriasis, rheumatoid arthritis, diabetes, muscular degeneration, bacterial infection, especially by Listeria monocytogenes and cancers.

The present invention accordingly provides, very particularly, for the use of the compounds I or II or formulation as defined above for preparing a medicament intended for the treatment or prevention of oncological diseases, and particularly intended for the treatment of cancers.

Among these cancers, a focus is on the treatment of solid or liquid tumours, and on the treatment of cancers which are resistant to cytotoxic agents.

The products of the present invention cited may in particular be used for the treatment of primary tumours and/or metastases, more particularly in gastric, hepatic, renal, ovarian, colonic, prostatic and lung (NSCLC and SCLC) cancers, glioblastomas, thyroid, bladder and breast cancers, in melanomas, in lymphoid or myeloid haematopoietic tumours, in sarcomas, in brain, larynx and lymphatic system cancers, and in bone and pancreatic cancers.

The present invention also provides for the use of the compounds I or II or formulation as defined above for preparing medicaments intended for the chemotherapy of cancers.

Medicaments of this kind intended for the chemotherapy of cancers may be used alone or in combination.

The products of the present application may in particular be administered alone or in combination with chemotherapy or radiotherapy or else in combination, for example, with other therapeutic agents.

Such therapeutic agents may be commonly used anti-tumour agents.

Kinase inhibitors include butyrolactone, flavopiridol and 2(2-hydroxyethylamino)-6- benzylamino-9-methylpurine, which is called olomoucine.

The present invention further provides, as new industrial products, the synthesis intermediates of formulae A1 , B1 , C1 , D1 and E1 as defined in the preparation of the examples above.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal or rectal administration, the sulphate salt may be administered in a unit administration form, as a mixture with conventional pharmaceutical excipients, to animals and to human beings for the prophylaxis or the treatment of the above disorders or diseases.

The appropriate unit administration forms include forms for oral administration such as tablets, soft or hard gel capsules, powders, granules and oral solutions or suspensions, and sublingual, buccal, intratracheal, intraocular and intranasal administration forms, forms for administration by inhalation, for topical, transdermal, subcutaneous, intramuscular or intravenous administration, rectal administration forms, and implants. For topical application, the compounds according to the invention may be used in creams, gels, ointments or lotions.

Said unit forms are dosed to allow daily administration of 0.01 to 20 mg of active principle per kg of body weight, depending on the formulation.

There may be special cases in which higher or lower doses are appropriate; such doses are not outside the scope of the invention. In accordance with customary practice, the dosage appropriate for each patient is determined by the physician according to the administration route and to the weight and response of said patient. The present invention, in another of its aspects, also relates to a method for treating the pathologies indicated above, which comprises administering to a patient an effective dose of sulphate.