MADRASIN-DERIVATIVE COMPOUNDS, COMPOSITION AND USES THEREOF FOR TREATING CANCER

Abstract

The present invention relates to the fields of medicine and in particular cancer treatment. The invention more specifically relates to new compounds which are typically for use as a medicament. In particular, the invention relates to the use of these new compounds for increasing the presentation, typically the production and presentation, of Pioneer Translation Products (PTPs)-derived antigens by cells, in particular cancer cells, or changing the immunopeptidome, in a subject, and inducing or stimulating an immune response in the subject. The present disclosure also relates to uses of such compounds, in particular to prepare a pharmaceutical composition and/or to allow or improve the efficiency of a therapy in a subject in need thereof. The invention also discloses methods for treating a disease, in particular cancer, for preventing or treating cancer metastasis and/or cancer recurrence, in a subject. The present invention in addition provides kits suitable for preparing a composition according to the present invention and/or for implementing the herein described methods.

Claims

1-15. (canceled)

16. A method for treating a disease in a subject, wherein the method comprises administering a subject in need thereof with a compound selected from ##STR00051## wherein: R.sup.1 and R.sup.2 are independently selected from H, CH.sub.4 and Cl, R.sup.3 is H, CH.sub.4, C.sub.2H.sub.6, n-Propyl, N(CH.sub.2).sub.4 or N(CH.sub.2).sub.5, Z is CH.sub.2 or C═O, n is 1, 2 or 3, and X is O, N or CH; ##STR00052## wherein: R.sup.1 and R.sup.2 are independently selected from H, CH.sub.4 (Methyl), Cl (Chlorine), and a thiosugar, and R.sup.3 is P(O)ONa).sub.2 or ##STR00053## wherein: R.sup.4 is H, CH.sub.4 (Methyl), C.sub.2H.sub.6(Ethyl), n-Propyl, N(CH.sub.2).sub.4 or N(CH.sub.2).sub.5, Z is CH.sub.2 or C═O, n is 1, 2 or 3, and X is O, N or CH; and N-(2-Methoxypyrimidin-5-yl)quinazolin-2-amine ##STR00054##

17. The method according to claim 16, wherein: the compound of formula (A) is 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one ##STR00055##  or the compound of formula (B) is selected from: Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate ##STR00056## N-(1H-Indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine ##STR00057## ((1S,2S,4R,5S,6S)-5-Acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ3,8λ3-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate ##STR00058## 7-Methoxy-4-meth 1-N-3,4,5-trimethoxyphenyl)quinazolin-2-amine ##STR00059## 7-Methoxy-N-(6-methoxypyrimidin-3-yl)-4-methylquinazolin-2-amine ##STR00060## N2-(7-Methoxy-4-methylquinazolin-2-yl)-N4-methoxypyrimidin-2,4-diamine ##STR00061## 7-Methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine ##STR00062##  and 7-Methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine ##STR00063##

18. The method according to claim 16, wherein the disease is cancer.

19. The method according to claim 18, wherein the compound is used in combination with at least one distinct anticancer agent and/or with radiotherapy.

20. The method according to claim 18, wherein cancer is selected from a carcinoma, sarcoma, lymphoma, germ cell tumor, blastoma, leukemia and multiple myeloma.

21. The method according to claim 20, wherein the compound is selected from the compound of formula IL, III, IV, V, VI and VII, and the cancer is a sarcoma.

22. The method according to claim 20, wherein the compound is selected from the compound of formula II, III, IV, V, VIII, IX, X and XI, and the carcinoma is a melanoma.

23. The method according to claim 19, wherein the at least one distinct anticancer agent is selected from a chemotherapeutic agent, an immune checkpoint blocker and an anti-cancer vaccine.

24. The method according to claim 18, wherein the compound stimulates an anti-cancer immune response in the subject.

25. The method according to claim 16, wherein the compound induces or increases the presentation or the production and presentation of Pioneer Translation Products (PTPs)-derived antigens by cancer cells, or changes the immunopeptidome, in the subject.

26. The method according to claim 16, wherein the subject is a mammal.

27. The method according to claim 26, wherein the mammal is a human being.

28. A composition comprising a compound according to claim 16 and a pharmaceutically acceptable carrier, optionally together with at least one distinct anticancer agent to be used simultaneously, separately or sequentially.

29. The composition according to claim 28, wherein: the compound of formula (A) is 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one ##STR00064##  or the compound of formula (B) is selected from: Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate ##STR00065## N-(1H-Indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine ##STR00066## ((1S,2S,4R,5S,6S)-5-Acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ3,8λ3-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate ##STR00067## 7-Methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine ##STR00068## 7-Methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine ##STR00069## N2-(7-Methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine ##STR00070## 7-Methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine ##STR00071##  and 7-Methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine ##STR00072##

30. A kit comprising a compound as described in claim 16 and at least one distinct anticancer agent in distinct containers.

Description

LEGENDS TO THE FIGURES

[0133] FIGS. 1A and 1B: Madrasin and Madra.HCl treatment increase intron-derived antigen presentation in cancer cells.

[0134] B3Z specific T-cell activation in MCA205 sarcoma cells and B16F10 melanoma cells expressing the intron-derived SL8 antigen after treatment with 5 μM or 10 μM (A) Madrasin (B) Madra.HCl. Free SL8 peptide was added in each condition to ensure that T-cell assays were carried out in nonsaturated conditions and that the expression of MHC-I molecules was taking into account in the results. Each graph is one representative of at least three independent experiments. Data are given as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired student t test).

[0135] FIG. 1C-1F: Madra.HCl slows down the growth of tumor bearing intron-derived SL8 epitope

[0136] Wild type MCA205 sarcoma cells or MCA205 sarcoma cells expressing the globin-SL8-intron construct (MCA205 globin-SL8-intron) were subcutaneously inoculated into the flank of immunocompetent C57BL/6 mice subsequently injected intraperitoneally with 20 mg/kg or 40 mg/kg of Madra.HCl at day 4, 7, 10 and 14 post tumor inoculation. Tumor size was assessed every 3 to 4 days until the established ethical endpoints were reached. The panel C represents the growth curve of MCA205 sarcoma cells expressing the globin-SL8-intron construct (MCA205 globin-SL8-intron), the panel D represents the tumor size at day 25. The panel E represents the growth curve of Wild type MCA205 sarcoma cells (MCA205 WT), the panel F represents the tumor size at day 21. Data are given as mean±SEM. *p<0.05, **p<0.01 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0137] FIG. 1G: Madra.HCl extends overall survival of mice bearing intron-derived SL8-expressing tumors

[0138] Kaplan-Meier survival curve of mice injected subcutaneously with MCA205 globin-SL8-intron and subsequently treated intraperitoneally with Madra.HCl at 20 mg/kg or 40 mg/kg, 4, 7, 10 and 14 days post tumor inoculation.

[0139] FIG. 1H: Madra.HCl induces a long-lasting specific antitumor response

[0140] Growth curve of MCA205 globin-SL8-intron cells and B16F10 WT cells inoculated at day 100 into the right and the left flank, respectively, of C57BL/6 mice which experienced complete tumor regression after treatment with Madra.HCl.

[0141] FIG. 2: Test MTT for each derivative of Madrasin

[0142] MTT assay performed on MCA205 (A) or B16F10 (B) cells treated with increase doses of the different derivatives of Madrasin. Data are expressed as half maximal inhibitory concentration (IC50) of the different derivatives. The IC50 represents the concentration of the tested compounds that is required for 50% inhibition of the cell viability compared to the control condition. A threshold of 60 μm is notified. It will be the maximum dose used for all the compounds that do not show any cell death.

[0143] FIG. 3: Derivatives of Madrasin that increase antigen presentation in both murine cell lines.

[0144] B3Z specific T-cell activation in MCA205 and B16F10 expressing the intron-derived SL8 antigen after treatment with 60 μM (A) of EYP59 (compound 7), 20 μM (B) of EYP201 (compound 6), 60 μM (C) of EYP165 (compound 57) or 50 μM (D) of EYP281 (compound 32). Free SL8 peptide was added in each condition to ensure that T-cell assays were carried out in nonsaturated conditions and that the expression of MHC-I molecules was taken into account in the results. Each graph is one representative of at least three independent experiments. Data are given as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired student t test).

[0145] FIG. 4: Derivatives of Madrasin that increase antigen presentation only in MCA205 sarcoma cell line.

[0146] B3Z specific T-cell activation in MCA205 expressing the intron-derived SL8 antigen after treatment with 60 μM (A) of EYP188 (compound 42), 50 μM (B) of EYP86 (compound 10). Free SL8 peptide was added in each condition to ensure that T-cell assays were carried out in nonsaturated conditions and that the expression of MHC-I molecules was taken into account in the results. Each graph is one representative of at least three independent experiments. Data are given as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired student t test).

[0147] FIG. 5: Derivatives of Madrasin that increase antigen presentation only in B16F10 melanoma cell line.

[0148] B3Z specific T-cell activation in MCA205 expressing the intron-derived SL8 antigen after treatment with 20 μM (A) of EYP174 (compound 41), 20 μM (B) of EYP179 (compound 50), 60 μM (C) of EYP190 (compound 54) and 10 μM (D) of EYP181 (compound 49). Free SL8 peptide was added in each condition to ensure that T-cell assays were carried out in nonsaturated conditions and that the expression of MHC-I molecules was taking into account in the results. Each graph is one representative of at least three independent experiments. Data are given as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired student t test).

[0149] FIG. 6: Derivatives of Madrasin that do not increase antigen presentation in both tumor cell lines.

[0150] B3Z specific T-cell activation in MCA205 expressing the intron-derived SL8 antigen after treatment with 60 μM (A) of EYP177 (compound 46), 60 μM (B) of EYP156 (compound 43), 10 μM (C) of EYP113 (compound 11) and 20 μM (D) of EYP102 (compound 9). Free SL8 peptide was added in each condition to ensure that T-cell assays were carried out in nonsaturated conditions and that the expression of MHC-I molecules was taking into account in the results. Each graph is one representative of at least three independent experiments. Data are given as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired student t test).

[0151] FIG. 7: EYP59 (compound 7) slows down the growth of MCA-WT sarcoma and MCA-intron-SL8.

[0152] Wild type MCA205 sarcoma cells or MCA205 sarcoma cells expressing the globin-SL8-intron construct (MCA205 globin-SL8-intron) were subcutaneously inoculated into the flank of immunocompetent C57BL/6 mice subsequently injected intraperitoneally with 20 mg/kg of EYP59 at day 4, 7, 10 and 14 post tumor inoculation. Tumor size was assessed every 3 to 4 days until the established ethical endpoints were reached (A). The upper panel B represents the growth curve of MCA205 sarcoma cells expressing the globin-SL8-intron construct (MCA205 globin-SL8-intron), the lower panel B represents the tumor size at day 23. The upper panel C represents the growth curve of Wild type MCA205 sarcoma cells (MCA205 WT), the lower panel C represents the tumor size at day 19. The panel D represents the growth curve of Wild type MCA205 sarcoma cells (MCA205 WT) in immunodeficient nu/nu mice with the same settings as previously described for the immunocompetent mice. Data are given as mean±SEM. *p<0.05, **p<0.01 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0153] FIG. 8: Madrasin derivative EYP59 (compound 7) efficiently reduces tumor growth in vivo when injected in intratumoral or intravenous ways.

[0154] Wild type MCA205 sarcoma cells were subcutaneously inoculated into the flank of immunocompetent C57BL/6 mice subsequently injected intratumorally with 2.5 mg/kg of EYP59 at day 4, 7, 10 and 14 post tumor inoculation or intravenously with 5 mg/kg of EYP59 at day 4, 7, 10 and 14 post tumor inoculation. The upper panel A represents the growth curve of wild type MCA205 sarcoma cells, the lower panel A represents the tumor size at day 21. The upper panel B represents the growth curve of wild type MCA205 sarcoma cell, the lower panel B represents the tumor size at day 25. Data are given as mean±SEM. *p<0.05, *p<0.01 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0155] FIG. 9: Synthesis of Madrasin and Madrasin hydrochloride (Madra.HCl).

[0156] FIG. 10: Pharmacomodulation of Madrasin and synthesis of Madrasin's derivatives.

EXAMPLES

[0157] Materials & Methods

[0158] Cell Culture

[0159] MCA205 mouse sarcoma cell line is cultured at 37° C. under 5% CO.sub.2 in RPMI 1640 medium (Life Technologies) in the presence of 1% glutamine, 1% sodium pyruvate, 1% non-essential amino-acids, 1% penicillin/streptomycin and 10% FBS (Life Technologies). B16F10 mouse melanoma cell line is cultured at 37° C. under 5% CO.sub.2 in DMEM medium (Life Technologies) containing 1% glutamine, 1% penicillin/streptomycin and 10% FCS under standard conditions. Stable MCA205-Globin-SL8-intron cell line are cultured under the same condition as MCA205 cell line with additional 500 μg/ml G418 (Life Technologies) for selection. Stable B16F10-Globin-SL8-intron cell line are cultured under the same condition as B16F10 cell line with additional 500 μg/ml G418 (Life Technologies) for selection. The SL8/Kb-specific (B3Z) T-cell reporter hybridoma are cultured at 37° C. under 5% CO.sub.2 in RPMI 1640 medium (Life Technologies) in the presence of 1% L-glutamine, 1% penicillin/streptomycin, 50 μM β-mercaptoéthanol and 10% FCS.

[0160] Schema of Synthesis of Madrasin and all the Derivatives Compounds

[0161] 1) Synthesis of Madrasin and Madrasin Hydrochloride (Madra.HCL)

##STR00026##

a. 7-Methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (Compound 1) (Ref. Org. Lett. 2015, 17, 4125)

[0162] ##STR00027##

[0163] Under argon, m-anisidine (2.3 mL, 20.3 mmol) and InCl.sub.3 (232 mg, 1.03 mmol) in acetone (30 mL) was heated at 50° C. for 14 h. The solvent was removed and the crude partitioned between DCM and aqueous saturated solution of Na.sub.2CO.sub.3. The organic layer was dried over MgSO.sub.4, filtered and concentrated. Purification by column chromatography (Cyclohexane/EtOAc 100:0->99:1) afforded the desired compound as a yellowish solid. The spectroscopic data are in accordance with the literature (Tamariz, J. et al. J. Org. Chem. 2013, 78, 9614-9626). Yield: 61% (2.51 g, 12.3 mmol). Mp 68.8° C. TLC Rf: 0.5 (Cyclo/EtOAc 9:1). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 6.97 (d, J=8.4 Hz, 1H), 6.20 (dd, J=8.4, 2.5 Hz, 1H), 6.01 (d, J=2.5 Hz, 1H), 5.19 (s, 1H), 3.75 (s, 3H), 1.96 (d, J=1.5 Hz, 3H), 1.26 (s, 6H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.13H.sub.18NO 204.1388, found 204.1385.

b. 1-(7-Methoxy-4-methylquinazolin-2-yl)guanidine (Compound 2)

[0164] ##STR00028##

[0165] 7-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (compound 1) (2.0 g, 0.98 mmol) and HCl (0.25 mL, 0.98 mmol, 4 M in dioxane) was stirred at room temperature for 30 min. Then H.sub.2O (95 mL) and dicyandiamide (831 mg, 0.98 mmol) was added and the mixture was refluxed for 48 h. After cooling at 60° C., the oil was filtered and the pH was adjusted to 11 with an aqueous saturated solution of NaHCO.sub.3. The precipitate formed was filtered off and dried in vacuo. The compound 2 was isolated as an off-white powder. Yield: 78% (1.76 g, 0.76 mmol). Mp 235-236° C. .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ 7.95 (d, J=9.1 Hz, 1H), 7.82 (s, 2H), 7.11 (d, J=2.5 Hz, 1H), 7.00 (dd, J=9.0, 2.5 Hz, 1H), 3.90 (s, 3H), 2.70 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.11H.sub.14N.sub.5O 232.1198, found 232.1193.

c. 2-((7-Methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (Madrasin, Compound 3)

[0166] ##STR00029##

[0167] A solution of 1-(7-methoxy-4-methylquinazolin-2-yl)guanidine (compound 2) (3.2 g, 13.8 mmol), ethyl 2-methyl-3-oxobutanoate (2.3 mL, 16.2 mmol), NaHCO.sub.3 (1.43 g, 17 mmol) in DMSO (22 mL) was heated at 110° C. for 48 h. After cooling at room temperature cold water was added. The precipitate formed was filtered off and purified by column chromatography (DCM/MeOH 100:0->98:2) to afford the madrasin 3 as a beige powder. Yield: 83% (3.58 g, 11.5 mmol). Mp 216.4-217.5° C. TLC Rf: 0.26 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 13.25 (s, 1H), 8.54 (s, 1H), 7.86 (d, J=9.1 Hz, 1H), 7.17 (d, J=2.5 Hz, 1H), 7.07 (dd, J=9.1, 2.5 Hz, 1H), 3.98 (s, 3H), 2.80 (s, 3H), 2.27 (s, 3H), 2.06 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.16H.sub.18N.sub.5O.sub.2312.1462, found 312.1458.

d. Madrasin Hydrochloride (Madr.HCl, EYP34)

[0168] ##STR00030##

[0169] To a solution of madrasin (compound 3) (600 mg, 1.98 mmol) in dry dioxane (10 mL) was added HCl (0.5 mL, 2 mmol, 4 M in dioxane). The mixture was allowed to stir at room temperature for 10 min. The precipitate formed was filtered off and dry in vacuo to afford the Madrasin chlorhydrate salt (565 mg, 1.68 mmol, 85%) as a clear green powder. .sup.1H NMR (300 MHz, Deuterium Oxide) δ 7.76 (s, 1H), 6.88 (s, 1H), 6.80 (s, 1H), 3.87 (s, 3H), 2.71 (s, 3H), 2.11 (s, 3H), 1.74 (s, 3H). Mp >330° C. (decomposition). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.16H.sub.18N.sub.5O.sub.2 312.1462, found 312.1460. Anal. Calcd for C.sub.16H.sub.18ClN.sub.5O.sub.2. 2/3H.sub.2O: C, 53.41; H, 5.42. Found: C, 53.40; H, 5.47.

[0170] 2) Pharmacomodulation of Madrasin

[0171] Part 1:

##STR00031##

a. 2-((7-Hydroxy-4-methylquinazoiin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (Compound 4, EYP107)

[0172] ##STR00032##

[0173] Under argon, at −78° C., to a solution of Madrasin (compound 3) (250 mg, 0.8 mmol) in DCE (15 mL) was added dropwise BBr.sub.3 (12 mL, 12 mmol). The solution was allowed to warm to room temperature and heated at 60° C. for 20 h. After quenching, the solvent was removed under reduced pressure. The crude was taken up with EtOAc and washed with an aqueous saturated solution of NaHCO.sub.3. The aqueous layer was extracted 10 times with EtOAc. The combined organic layers were dried over MgSO.sub.4, filtered and concentrated. The desired compound 4 was precipitate in DCM, filtered and washed several times with DCM. Yield: 42% (1.0 g, 0.34 mmol). Mp 348.8-349.9° C. TLC Rf: 0.18 (DCM/MeOH 97:3). .sup.1H NMR (300 MHz, DMSO) δ 13.52 (s, 1H), 10.93 (s, 2H), 8.05 (d, J=9.0 Hz, 1H), 7.03 (dd, J=9.8, 1.1 Hz, 1H), 6.94 (d, J=2.2 Hz, 1H), 2.77 (s, 3H), 2.18 (s, 3H), 1.90 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.15H.sub.16N.sub.5O.sub.2298.1304, found 298.1312.

b. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methyl quinazolin-7-olate (Compound 5, EYP112)

[0174] ##STR00033##

[0175] To a suspension of 2-((7-hydroxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (compound 4) (15 mg, 0.05 mmol) in water (1.5 mL) was added NaOH (50 μL, 0.05 mmol, 1 M in H.sub.2O). The stirring was continued for 5 minutes until a clear solution was obtained. After evaporation to dryness, a yellow solid was obtained. Yield: >99% (15 mg, 0.05 mmol). .sup.1H NMR (300 MHz, D.sub.2O) δ 7.51 (d, J=8.8 Hz, 1H), 6.67 (d, J=8.8 Hz, 1H), 6.40 (s, 1H), 2.49 (s, 3H), 2.03 (s, 3H), 1.71 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.15H.sub.16N.sub.5O.sub.2 298.1304, found 298.1259.

c. 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (Compound 6, EYP201)

[0176] ##STR00034##

[0177] Under an inert atmosphere, compound (5 mg, 0.11 mmol) was suspended in DMF (1 mL) and KOH (13 mg, 0.23 mmol) was added. The mixture was stirred at room temperature for 1 h until it became clear. Then 2-chloro-N-ethylmorpholine (22 mg, 0.12 mmol) was added and the mixture was stirred for 14 h at room temperature. The solvent was removed in vacuo. The compound 6 was obtained after column chromatography (DCM/MeOH 100:0->97:3) as a off-white powder. Yield: 19% (9.1 mg, 0.022 mmol). TLC Rf: 0.2 (DCM/MeOH 96:4). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 13.26 (s, 1H), 7.85 (d, J=9.0 Hz, 1H), 7.19 (s, 1H), 7.08 (d, J=9.0 Hz, 1H), 4.29 (t, J=5.6 Hz, 2H), 3.76 (t, J=4.7 Hz, 4H), 2.89 (t, J=5.7 Hz, 2H), 2.80 (s, 3H), 2.62 (t, J=4.6 Hz, 4H), 2.26 (s, 3H), 2.05 (s, 3H). HRMS (ESI) (M+H)+m/z calculated for C.sub.21H.sub.27N.sub.6O.sub.3 411.2145, found 411.2152.

d. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (Compound 7, EYP59)

[0178] ##STR00035##

[0179] A suspension of compound 4 (100.4 mg, 0.33 mmol) and KOH (25 mg, 0.44 mmol) in H.sub.2O (5 mL) was stirred at room temperature for 30 min. DCM (5 mL) was then added, followed by (EtO).sub.2P(O)Cl (0.064 mL, 0.44 mmol) and TBAB (141 mg, 0.44 mmol). The mixture was stirred at room temperature for 2 h. The organic layer was separated and the aqueous layer was extracted with DCM. The combined organic layers were dried over MgSO.sub.4, filtered and concentrated in vacuo. The intermediate diethylphosphate derivative was obtained after column chromatography (DCM/MeOH 100:0->97:3). Yield: 38% (55 mg, 0.12 mmol). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 13.16 (s, 1H), 7.98 (d, J=9.0 Hz, 1H), 7.66 (d, J=2.3 Hz, 1H), 7.42 (dd, J=9.0, 2.4 Hz, 1H), 4.28 (p, J=7.4 Hz, 4H), 2.84 (s, 3H), 2.27 (s, 3H), 2.05 (s, 3H), 1.39 (t, J=7.0 Hz, 6H).

[0180] The purified diethylphosphate (55 mg, 0.12 mmol) compound was then solubilized in DCM (2.5 mL); TMSI was added and the reaction was stirred for 3 h at room temperature. The solvent was evaporated and the crude was triturated with DCM, filtered and then was suspended in H.sub.2O. An aqueous solution of NaOH (0.250 mL, 0.254 mmol) was slowly added and the mixture became limpid after 30 min of stirring. The solvent was removed under vacuum to afford Madrasin-phosphate (compound 7). Yield: 93% (50 mg, 0.11 mmol). .sup.1H NMR (300 MHz, D.sub.2O) δ 7.97 (d, J=9.1 Hz, 1H), 7.47-7.38 (m, 2H), 2.73 (s, 3H), 2.26 (s, 3H), 1.87 (s, 3H). .sup.31P NMR (81 MHz, D.sub.2O) δ −174.17. HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.15H.sub.16N.sub.5NaO.sub.5P 400.0787, found 400.0792.

[0181] Part 2:

##STR00036##

a. 2-((8-Iodo-7-methoxy-4-methylquinazolin-2-yl)amino-5,6-dimethylpyrimidin-4(3H)-one (Compound 8)

[0182] ##STR00037##

[0183] To a cooled solution of Madrasin (compound 3) (360 mg, 1.16 mmol) in H.sub.2SO.sub.4 conc. (1.2 mL) was added (in the dark) NIS (248 mg, 1.10 mmol). The solution was stirred at room temperature for 5 h. The mixture was poured in cold water and the pH was adjusted to 5. After extraction with DCM, the organic layers combined were dried over MgSO.sub.4, filtered and concentrated. Purification by column chromatography (DCM/MeOH 100:0->98:2) afforded the desired compound 8. Yield: 25% (127 mg, 0.29 mmol). TLC Rf: 0.22 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 13.50 (s, 1H), 8.88 (s, 1H), 7.96 (d, J=9.1 Hz, 1H), 7.09 (d, J=9.1 Hz, 1H), 4.08 (s, 3H), 2.83 (s, 3H), 2.28 (s, 3H), 2.07 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.16H.sub.17N.sub.5O.sub.2438.0427, found 438.0427.

b. N-Acetyl-S-(2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)cysteine (Compound 9, EYP102)

[0184] ##STR00038##

[0185] In a sealable vial, under an inert atmosphere, were added 2-((8-iodo-7-methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one compound 8 (10.6 mg, 0.025 mmol), cysteineNHAc (7 mg, 0.04 mmol), PdG.sub.3Xantphos (5 mg, 0.005 mmol) and THF (0.1 mL). The mixture was purged with argon and NEt.sub.3 (10 μL, 0.07 mmol) was added. The mixture was stirred at 50° C. for 18 h and at 70° C. for 6 h. The solvent was removed and purification by column chromatography (DCM/MeOH 100:0->70:30) afforded the desired compound 9. A second purification by preparative TLC was necessary (DCM/MeOH 75:25) to furnish pure compound 9 as a white powder. Yield: 48% (5.7 mg, 0.012 mmol). TLC Rf: 0.1 (DCM/MeOH 8:2). .sup.1H NMR (300 MHz, MeOD) δ 8.11 (d, J=9.1 Hz, 1H), 7.32 (d, J=9.0 Hz, 1H), 4.08 (s, 3H), 3.70-3.55 (m, 2H), 2.80 (s, 3H), 2.70 (sl, 1H), 2.23-1.98 (m, 6H), 1.73 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.21H.sub.25N.sub.6O.sub.5S 473.1607, found 473.1608.

c. ((1S,2S,4R,5S,6S)-5-Acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ.SUP.3.,8λ.SUP.3.-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate (Compound 10, EYP86)

[0186] ##STR00039##

[0187] Under an inert atmosphere, a solution of 2-((8-iodo-7-methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one compound 8 (25 mg, 0.055 mmol) and thioglucose (22 mg, 0.06 mmol) in THF (0.5 mL) was purged with argon. The catalyst PdG.sub.3Xantphos (10 mg, 0.01 mmol) and Et.sub.3N (20 μL, 0.14 mmol) were added and the reaction mixture were heated at 50° C. for 3 h. The solvent was removed in vacuo and purification by column chromatography (DCM/MeOH 100:0->97:3) afforded the desired compound 10 as an orange solid. Yield: 41%. (15.4 mg, 0.022 mmol). TLC Rf: 0.3 (DCM/MeOH 96:4). .sup.1H NMR (300 MHz, MeOD) δ 8.18 (d, J=9.1 Hz, 1H), 7.34 (d, J=9.5 Hz, 1H), 5.17 (t, J=9.3 Hz, 1H), 4.75-4.66 (m, 1H), 4.07 (s, 3H), 3.99-3.89 (m, 1H), 3.62-3.52 (m, 2H), 2.83 (s, 3H), 2.62 (s, 2H), 2.10 (s, 6H), 1.94 (d, J=9.4 Hz, 6H), 1.66 (s, 3H). HRMS (ESI) (M+Na).sup.+ m/z calculated for C.sub.30H.sub.35N.sub.5O.sub.11NaS 696.1651, found 696.1657.

d. 2-((7-Methoxy-4-methyl-8-(((2R,3S,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)quinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (Compound 11, EYP113)

[0188] ##STR00040##

[0189] A solution of compound 10 in MeONa (0.19 mL, 0.038 mmol, 0.2 M in MeOH) was stirred at room temperature for 1 h. After evaporation of the solvent in vacuo, compound 11 was obtained as a yellow powder. Yield: >99% (7 mg, 0.014 mmol). .sup.1H NMR (300 MHz, MeOD) δ 7.88 (d, J=9.0 Hz, 1H), 7.02 (d, J=9.1 Hz, 1H), 3.87 (s, 3H), 3.53-3.28 (m, 5H), 2.98-2.91 (m, 2H), 2.58 (s, 2H), 2.10 (s, 3H), 1.80 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.22H.sub.28N.sub.5O.sub.7S 506.1709, found 506.1729.

[0190] 3) General Procedure A:

##STR00041##

[0191] Under an inert atmosphere, were added 2-aminoquinazoline derivative (1 equiv) (compound 12), aryl halide (1.5 equiv; unless otherwise stated), Pd.sub.2dba.sub.3 (7.5 mol % or 10 mol %), Xantphos (15 mol % or 20 mol %) and Cs.sub.2CO.sub.3 (2 equiv). The mixture was purged with argon. THF was added. The vial was sealed and the reaction mixture was stirred at 80° C. for 14 h. After cooling at room temperature, the solvent was removed under vacuum and the crude was purified by column chromatography to afford the desired compound.

[0192] N-(2-Methoxypyrimidin-5-yl)quinazolin-2-amine (Compound 32, EYP281, C.sub.13H.sub.11N.sub.5O, 253,2650 g/mol): Following the general procedure A, starting from 2-aminoquinazoline (21 mg, 0.13 mmol), 5-bromo-2-methoxypyrimidine (26 mg, 0.19 mmol), Pd.sub.2dba.sub.3 (9 mg, 0.01 mmol), Xantphos (12 mg, 0.02 mmol), Cs.sub.2CO.sub.3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 42% (13 mg, 0.05 mmol). Beige powder. Mp 210.9-211.7° C. TLC Rf: 0.2 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 9.11 (s, 1H), 9.03 (s, 2H), 7.77 (d, J=7.4 Hz, 3H), 7.38 (t, J=7.3 Hz, 1H), 7.21 (s, 1H), 4.04 (s, 3H).

[0193] 4) Another Series from the Methodology Part

##STR00042##

a. 7-Methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (Compound 41, EYP174)

[0194] ##STR00043##

[0195] Following the general procedure A (except that only 1.1 equiv of heteroaryl bromide were used), starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 2-methoxy-5-bromopyridine (0.017 mL, 0.14 mmol), Pd.sub.2dba.sub.3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs.sub.2CO.sub.3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 76% (29 mg, 0.10 mmol). Orange powder. Mp 172.1-173.0° C. TLC Rf: 0.6 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 8.59 (d, J=2.8 Hz, 1H), 8.04 (dd, J=8.8, 2.8 Hz, 1H), 7.75 (d, J=9.1 Hz, 1H), 7.36 (s, 1H), 6.98 (d, J=2.5 Hz, 1H), 6.89 (dd, J=9.0, 2.5 Hz, 1H), 6.76 (d, J=8.8 Hz, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 2.72 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.16H.sub.17N.sub.4O.sub.2297.1352, found 297.1328.

b. 7-Methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (Compound 42, EYP188)

[0196] ##STR00044##

[0197] Following the general procedure A, starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39)(25 mg, 0.13 mmol), 1-bromo-3,4,5-trimethoxybenezene (47 mg, 0.19 mmol), Pd.sub.2dba.sub.3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs.sub.2CO.sub.3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 58% (26 mg, 0.07 mmol). Pale yellow powder; Mp 159.5-160.1° C. TLC Rf: 0.8 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 7.78 (d, J=9.0 Hz, 1H), 7.39 (s, 1H), 7.11 (s, 2H), 6.97 (d, J=2.6 Hz, 1H), 6.92 (dt, J=9.0, 2.4 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 6H), 3.83 (s, 3H), 2.75 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.19H.sub.22N.sub.3O.sub.4 356.1610, found 356.1546.

c. 7-Methoxy-4-methyl-N-(p-tolyl)quinazolin-2-amine (Compound 43, EYP156)

[0198] ##STR00045##

[0199] Following the general procedure A, starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 4-bromotoluene (32 mg, 0.19 mmol), Pd.sub.2dba.sub.3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs.sub.2CO.sub.3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 39% (14.0 mg, 0.05 mmol). Off-white solid. Mp 142.8-143.3° C. TLC Rf: 0.7 (DCM 100%). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 7.77 (d, J=9.0 Hz, 1H), 7.68 (d, J=8.2 Hz, 2H), 7.24-7.12 (m, 3H), 7.05 (d, J=2.5 Hz, 1H), 6.90 (dd, J 10=9.1, 2.5 Hz, 1H), 3.93 (s, 3H), 2.75 (s, 3H), 2.34 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.17H.sub.18N.sub.3O 280.1450, found 280.1444.

d. t-Butyl 3-((7-methoxy-4-methylquinazolin-2-yl)amino)-1H-indole-1-carboxylate (Compound 46, EYP177)

[0200] ##STR00046##

[0201] Following the general procedure A (except that only 1.1 equiv of heteroaryl bromide were used), starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 3-bromoindole derivative (41 mg, 0.14 mmol), Pd.sub.2dba.sub.3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs.sub.2CO.sub.3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 31% (16 mg, 0.04 mmol). Orange powder. Mp 114-116° C. TLC Rf: 0.55 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 8.57 (s, 1H), 8.17 (s, 1H), 7.82 (d, J=9.0 Hz, 1H), 7.58 (d, J=7.7 Hz, 1H), 7.40-7.27 (m, 4H), 7.09 (d, J=2.5 Hz, 1H), 6.94 (dd, J=9.1, 2.5 Hz, 1H), 3.95 (s, 3H), 2.80 (s, 3H), 1.73 (s, 9H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.23H.sub.25N.sub.4O.sub.3 405.1927, found 405.1932.

e. N.SUP.2.-(7-Methoxy-4-methylquinazolin-2-yl)-N.SUP.4.-methylpyrimidine-2,4-diamine (Compound 49, EYP181)

[0202] ##STR00047##

[0203] Following the general procedure A (except that only 1.1 equiv of heteroaryl chloride were used), starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 2-chloro-N-methylpyrimidin-4-amine (20 mg, 0.14 mmol), Pd.sub.2dba.sub.3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs.sub.2CO.sub.3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 31% (11 mg, 0.04 mmol). Grey powder. Mp 247.4-249.1° C. TLC Rf: 0.45 (DCM/MeOH 9:1). .sup.1H NMR (300 MHz, MeOD) δ 8.02 (d, J=9.2 Hz, 2H), 7.49 (s, 1H), 7.13 (d, J=9.2 Hz, 1H), 6.39 (d, J=6.5 Hz, 1H), 4.00 (s, 3H), 3.07 (s, 3H), 2.85 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.15H.sub.17N.sub.6O 297.1464, found 297.1473.

f. 7-Methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (Compound 50, EYP179)

[0204] ##STR00048##

[0205] Following the general procedure A (except that only 1.1 equiv of heteroaryl chloride were used), starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 2-chloro-4-methoxypyrimidine (20 mg, 0.14 mmol), Pd.sub.2dba.sub.3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs.sub.2CO.sub.3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 62% (23 mg, 0.08 mmol). Yellow powder. Mp 160.8-161.5° C. TLC Rf: 0.3 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 8.42-8.33 (m, 1H), 8.28-8.16 (m, 1H), 7.84 (d, J=9.0 Hz, 1H), 7.23 (s, 1H), 7.01 (dd, J=9.0, 2.3 Hz, 1H), 6.35 (d, J=5.6 Hz, 1H), 3.97 (s, 3H), 3.92 (s, 3H), 2.81 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.15H.sub.16N.sub.5O.sub.2298.1304, found 298.1302.

g. 7-Methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (Compound 54, EYP190)

[0206] ##STR00049##

[0207] To a solution of compound 53 (EYP193) (10 mg, 0.033 mmol) in MeOH (0.5 mL) was added NaOMe (0.15 mL, 0.075 mmol). The mixture was heated at 80° C. for 6 h. The solvent was then removed in vacuo and purification by column chromatography (DCM/MeOH 100:0->99:1) afforded the desired compound 54 as a white solid. Yield: 61% (6 mg, 0.02 mmol). Mp 173.9-174.3° C. TLC Rf: 0.27 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 8.41 (d, J=5.7 Hz, 1H), 8.35 (d, J=5.8 Hz, 1H), 7.96 (s, 1H), 7.86 (d, J=9.0 Hz, 1H), 7.12 (d, J=2.6 Hz, 1H), 7.05 (dd, J=9.1, 2.5 Hz, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 2.81 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.15H.sub.16N.sub.5O 2298.1304, found 298.1309.

h. N-(1H-Indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (Compound 57, EYP165)

[0208] ##STR00050##

[0209] To a solution of compound 46 (EYP177) (35 mg, 0.08 mmol) in DCM (2 mL) was added TFA (0.1 mL, 1.3 mmol). The solution was stirred at room temperature for 2 h. After addition of an aqueous saturated solution of NaHCO.sub.3 and extraction with DCM, the organic layer was dried over MgSO.sub.4, filtered and concentrated to dryness. Purification by column chromatography (DCM/MeOH 100:1->200:1) afforded the desired compound 57 as a white solid. Yield: 48% (11 mg, 0.04 mmol). White powder. TLC Rf: 0.6 (DCM/MeOH 98:2). .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ 10.69 (s, 1H), 9.51 (s, 1H), 8.17 (s, 1H), 8.01 (d, J=8.0 Hz, 1H), 7.91 (d, J=8.9 Hz, 1H), 7.34 (d, J=8.1 Hz, 1H), 7.09 (t, J=7.5 Hz, 1H), 7.01 (d, J=2.4 Hz, 1H), 6.96 (t, J=7.5 Hz, 1H), 6.88 (dd, J=9.0, 2.5 Hz, 1H), 3.91 (s, 3H), 2.73 (s, 3H). HRMS (ESI) (M+H).sup.+ m/z calculated for C.sub.18H.sub.17N.sub.4O 305.1402, found 305.1408.

[0210] T-Cell Assay

[0211] MCA205 and B16F10 mouse cell lines are transfected with the plasmid YFP-Globin-SL8-intron or with the PCDNA3 empty plasmid (negative control) with the transfection reagent jetPRIME (Ozyme) or GeneJuice (Millipore) respectively according to each manufacturer protocol. Twenty-four hours after transfection, cells are treated with different doses of Madrasin (Sigma SML 1409), Madra.HCl (3.HCl) or the different compounds herein cited. Cells are then washed three times with PBS 1× and 5.Math.10.sup.4 tumor cells are co-cultured with 1.Math.10.sup.5 B3Z hybridoma in a 96-well plate. In positive control wells, 4 μg/ml of synthetic peptide SL8 is added. Cells are then incubated overnight at 37° C. with 5% CO.sub.2. The plate is centrifuged at 1200 rpm for 5 min, cells are washed twice with PBS 1× and lysed for 5 min at 4° C. under shaking in 0.2% TritonX-100, 0.2% DTT, 0.5M K.sub.2HPO.sub.4, 0.5M KH.sub.2PO.sub.4. The lysate is centrifuged at 3000 rpm for 10 min and the supernatant is transferred to a 96-well optiplate (OptiPlaque-96, PerkinElmer). A revelation buffer containing 10 mM MgCl.sub.2, 11.2 mM β-mercaptoethanol, 0.0015% IGEPAL® CA-630 and 40 μM 4-Methylumbelliferyl β-D-Galactopyranoside (MUG) in PBS is added and the plate is incubated at room temperature for 3 hours. Finally, the β-galactosidase activity is measured using the FLUOstar OPTIMA (BMG LABTECH Gmbh, 30 Offenburg, Germany). Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired student t test).

[0212] Tumor Challenge and Treatment

[0213] C57Bl/6J female mice are obtained from Harlan Laboratories Ltd, Switzerland. 7 week-old mice are injected subcutaneously into the right flank with 1.Math.10.sup.5 MCA205 sarcoma or B16F10 melanoma cells or 1, 5.Math.10.sup.5 MCA205 globin-SL8-intron or B16F10 globin-SL8-intron cells. Sarcoma cells are injected in 100 μL sterile PBS while melanoma cells are injected in 50:50 PBS:Matrigel to prevent tumor cell dissemination. Madra.HCl is injected 4, 7, 10 and 14 days post tumor inoculation, in 100 μL H.sub.2O+30% (2-Hydroxypropyl)-β-cyclodextrin (w/v). In the same manner EYP59 (compound 7) is injected 4, 7, 10 and 14 days post tumor inoculation, in 100 μL H.sub.2O. Area of the tumor is recorded every 3 to 4 days until the tumor reaches the ethical end points. All animal experiments were carried out in compliance with French and European laws and regulations. Results are expressed as mean±SEM. *p<0.05, **P<0.01, ***P<0.001 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0214] Results

[0215] Madrasin Treatment Increases Antigenic Presentation of PTP-Derived Antigens in Cancer Cells.

[0216] In recent studies inventors have shown that PTPs is a major source of peptides for the endogenous MHC class I pathway in vitro. In order to modulate the presentation PTPs-derived antigens at cancer cells surface, inventors tested the impact of Madrasin treatment on mice tumor cell lines, one melanoma (B16F10) and one sarcoma (MCA205) cell lines. Both murine cell lines were transiently expressing the PTPs-SL8 epitope derived from an intron within the β-Globin gene construct or were kept untransfected. The Madrasin elicits an increase in the PTPs-dependent antigen presentation, with a dose dependent effect in both mouse cell lines (FIG. 1A). These results show that production and presentation of PTPs-derived antigens can be positively modulated in cancer cell lines upon Madrasin treatment. They support the hypothesis that this molecule could be used as positive immunomodulator to potentiate a specific anti-tumoral immune response dependent on the PTPs production and presentation.

[0217] Synthesized Madrasin Hydrochloride (Madra.HCl) Treatment Increases Antigenic Presentation of PTP-Derived Antigen In Vitro and Slow Down Tumor Growth In Vivo.

[0218] The above results demonstrate that the Madrasin is able to increase the production and presentation in vitro of PTPs-dependent antigen encoded by intron sequences at the cell surface of treated tumor cell lines. The next question was to see if the Madrasin, which can be only dissolved in DMSO, can have the same effect on tumor growth and CD8.sup.+ T cell proliferation in vivo. Unfortunately, inventors were not able to perform an in vivo experiment with Madrasin because of its week concentration in DMSO. As this first result was encouraging, they decided to generate derivatives of the Madrasin in order to increase cancer immune responses. In fact, Madrasin is insoluble in water and can only be dissolved in DMSO solvent rendering its pharmacokinetic in mice less efficient.

[0219] In order to make Madrasin available for broader in vitro and in vivo validation without the use of toxic carriers or cosolvents (DMSO), it was considered necessary to find a strategy to synthesize Madrasin and some derivatives to enhance their solubility and immunomodulator activities.

[0220] To this end phosphate prodrugs for example can typically display excellent water solubility, chemical stability, and rapid bioconversion back to the parent drug by phosphatases. The formation of phosphate prodrugs has been applied to increase the aqueous solubility of a variety of molecules, including antineoplastic phenolic natural products and their derivatives, exemplified by combretastatin A-4.

[0221] In view to test the news compounds as positive immunomodulator against tumor cell lines, inventors first decided to test them in an in vitro assay. As expected, the new compound, herein identified as “Madra.HCl” was able to be dissolved in water. After treatment of both murine cell lines MCA205 or B16F10 transiently expressing PTPs-SL8 epitope derived from an intron in the β-Globin gene (Globin-intron-SL8) with 5 μM or 10 μM of Madra.HCl, inventors observed an increase in PTPs-dependent antigen presentation in a dose dependent manner (FIG. 1B). 1.Math.10.sup.5 MCA205 sarcoma cells stably expressing the SIINFEKL (SL8) epitope from an intron in the β-Globin gene (Globin-intron-SL8) were subcutaneously inoculated in mouse. Four days after this inoculation, the mice were intraperitoneally vaccinated with a define dose of Madra.HCl. Then every 3 days 20 or 40 mg/kg of Madra.HCl were injected. During that time the tumor growth was monitored every two to three days (FIG. 1C).

[0222] Inventors observed a significant 50% reduction of tumor growth at day 27 after challenge in mice treated with 20 mg/kg of Madra.HCl (FIG. 1D) and a further 60 to 70% decrease of tumor growth at day 27 after challenge in mice treated with 40 mg/kg of Madra.HCl (FIG. 1D). The same experiment is performed on B16F10 tumor cells expressing the Globin-intron-SL8 construct.

[0223] In the same manner, 1.Math.10.sup.5 untransfected MCA sarcoma cells were subcutaneously inoculated in mouse.

[0224] Four days after this inoculation, the mice were intraperitoneally vaccinated with a define dose of Madra.HCl. Then, every 3 days the same doses were again injected. During that time the tumor growth was monitored every two to three days (FIG. 1E). Inventors observed a significant 40% reduction of tumor growth at day 21 after challenge in mice treated with 20 mg/kg or 40 mg/kg of Madra.HCl (FIG. 1F).

[0225] Furthermore, Madra.HCl treatment was shown to extend survival of mice, with around 25% of survivors 120 days after tumor inoculation when mice were treated with 40 mg/kg of Madra.HCl and with around 15% of survivors 120 days after tumor inoculation when mice were treated with 20 mg/kg of Madra.HCl (FIG. 1G).

[0226] Finally, mice which were inoculated with MCA205 tumor cells expressing the Globin-intron-SL8 construct, and that experienced complete tumor regression after treatment with Madra.HCl as described above were re-challenged 100 days later with MCA205 tumor cells expressing the Globin-intron-SL8 construct on the right flank and with B16F10 tumor cells on the left flank. While B16F10 tumors grew over time, the MCA205 tumor cells did not grow in mice (FIG. 1H). These results demonstrate that mice developed a long term anti-tumoral response specific to MCA205 tumor after Madra.HCl treatment.

[0227] Madrasin Derivatives Efficiently Increase MHC Class I Presentation of Intron-Derived Antigen In Vitro.

[0228] In order to improve the immunomodulatory activity of the Madra.HCl for broader in vitro and in vivo validation without the use of toxic carriers or cosolvents (DMSO), it was considered necessary to find a strategy to change its structure by steal keeping its activity. The next compounds of the present invention were prepared as herein above described.

[0229] In order to test the new compounds as positive immunomodulators against tumor cell lines, inventors first decided to test them in an in vitro assay. As expected, few derivatives of Madra.HCl were soluble in water and some other not. Using an MTT test, they identified the IC25 and the IC50 for each compounds in MCA sarcoma and B16F10 melanoma cell lines (FIG. 2). For the rest of the experiments, they decided to treat both cell lines with the IC50 of each compound soluble or not in water. For that purpose, both murine tumor cell lines, transiently expressing PTPs-SL8 epitope derived from an intron in the β-Globin gene (Globin-intron-SL8), were treated O/N with each compounds. Four different observations for the immunomodulatory activity of each compound were made: The first was that, they noticed an increase in PTPs-dependent antigen presentation with some of the compounds in both murine tumor cell lines (FIG. 3). They could see an increase of the PTP-SL8 presentation after treatment of the murine cell lines with EYP59 (compound 7) (A), EYP201 (compound 6) (B), EYP165 (compound 57) (C) and EYP281 (compound 32) (D) respectively.

[0230] The second observation was that some compounds were able to positively increase presentation of their PTPs-derived antigen in only MCA cell lines transiently expressing PTPs-SL8 epitope derived from an intron in the β-Globin gene (FIG. 4). They could see an increase of the PTP-SL8 presentation after treatment of the murine sarcoma cell lines with EYP188 (compound 42) (A) and EYP86 (compound 10) (B) respectively.

[0231] Furthermore, the third observation was that some compounds were able to positively increase presentation of our PTPs-derived antigen in only B16F10 cell lines transiently expressing PTPs-SL8 epitope derived from an intron in the β-Globin gene (FIG. 5). Inventors could see an increase of the PTP-SL8 presentation after treatment of the murine melanoma cell lines with EYP174 (compound 41) (A), EYP179 (compound 50) (B), EYP190 (compound 54) (C) and EYP181 (compound 49) (D) respectively.

[0232] And finally, the fourth observation was that some compounds were not able to positively increase the presentation of our PTPs-derived antigen in both murine cell lines tested (FIG. 6). Inventors could not see any increase of the PTP-SL8 presentation after treatment of both murine cell lines with EYP177 (compound 46)(A), EYP156 (compound 43)(B), EYP113 (compound 11) (C), and EYP102 (compound 9) (D) respectively.

[0233] Madrasin Derivative EYP59 (Compound 7) Efficiently Reduces Tumor Growth In Vivo in an Immune-Dependent Manner.

[0234] 1.Math.10.sup.5 MCA205 sarcoma cells stably expressing the SIINFEKL (SL8) epitope from an intron in the β-Globin gene (Globin-intron-SL8) were subcutaneously inoculated in mouse. Four days after this inoculation, the mice were intraperitoneally vaccinated with a define dose of EYP59. Then, every 3 days, 20 mg/kg were injected. During that time the tumor growth was monitored every two to three days (FIG. 7A). Inventors observed a significant 85% reduction of tumor growth at day 23 after challenge in mice treated with 20 mg/kg of EYP59 (FIG. 7B). In the same manner, 1.Math.10.sup.5 untransfected MCA205 sarcoma cells were subcutaneously inoculated in mouse. Four days after this inoculation, the mice were intraperitoneally vaccinated with a define dose of EYP59. Then, every 3 days the same doses were again injected. During that time, the tumor growth was monitored every two to three days. Inventors observed a significant 70% reduction of tumor growth at day 19 after challenge in mice treated with 20 mg/kg of EYP59 (FIG. 7C).

[0235] In order to assess the requirement of the immune response for this effect, inventors tested the impact of 20 mg/kg EYP59 treatment in immunodeficient nu/nu mice with the same settings as previously described and observed that it has no effect on the tumor growth (FIG. 7D). These results show that tumor size reduction upon EYP59 treatment requires the presence of an active immune response in vivo.

[0236] Madrasin Derivative EYP59 (Compound 7) Efficiently Reduces Tumor Growth In Vivo when Injected in Intratumoral or Intravenous Ways.

[0237] 1.Math.10.sup.5 wild type MCA205 sarcoma cells were subcutaneously inoculated in mouse. Four days after this inoculation, the mice were injected with a define dose of EYP59 intratumorally (2.5 mg/kg) or intravenously (5 mg/kg). Then, every 3 days, the same amount of product was injected respectively of the way of administration. During that time the tumor growth was monitored every two to three days.

[0238] Inventors observed a significant 75% reduction of tumor growth at day 21 after challenge in mice treated intratumorally with 2.5 mg/kg of EYP59 (FIG. 8A). Furthermore, Inventors observed a significant 80% reduction of tumor growth at day 25 after challenge in mice treated intravenously with 5 mg/kg of EYP59 (FIG. 8B).

DISCUSSION

[0239] The present invention reveals that specific spliceosome inhibitors have a positive effect on the antitumor immune response and therefore on tumor growth. Splicing abnormalities have emerged as a specific feature of cancer and are studied as predictive markers for patient survival as well as targets for cancer treatments with splicing inhibitors, some of which are currently in development in acute myeloid leukemia. In the present invention inventors demonstrate that some specific derivatives of Madrasin are potent stimulators of the anti-tumor immune response in vitro in sarcoma and melanoma tumor cell lines. They also demonstrate that some Madrasin derivatives are specifically increasing the PTP-dependent antigen presentation in sarcoma cancer model and that some others, different from the first set, are also capable to increase the FTP-dependent antigen presentation in melanoma cancer model. They open the way to new applications within the framework of targeted molecular therapies by highlighting original biomolecular profiles.

[0240] The PTPs model describes the pre-spliced mRNA as the template for PTP by an alternative translational event occurring in the nucleus. In the study describing this alternative translation, inventors demonstrated that forced nuclear retention of the mRNA encoding the intronic SL8 peptide leads to an increase in the SL8 antigen presentation. Besides, pladienolides and spliceostatin A (SSA) have been shown to inhibit the splicing by targeting the SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein (snRNP) in the spliceosome, and have been described to promote pre-RNA accumulation in the nucleus. The Madrasin inhibits differently the splicing by preventing stable U4/U61U5 tri-snRNPs recruitment right after the U2 snRNP fixation. However, it is likely that it also induces pre-mRNA accumulation in the nucleus, resulting in the increase in antigen presentation. The link between pre-mRNA nuclear accumulation and increased antigen presentation is not known. It is tempting to hypothesize that pre-mRNA accumulation in the nucleus provides more templates for PTP production leading to the enrichment of SL8-containing PTPs, used as a major source for SL8 direct presentation.

[0241] Furthermore, pre-mRNA splicing is an essential mechanism required for the normal function of all mammalian cells. In the last few years, several studies reported the presence of mutations and overexpression of main spliceosome factors associated with aberrant splicing activity in various cancers. Few years ago, inventors have also provided some evidence that the inhibition of the spliceosome increases MHC class I PTPs-dependent antigen presentation. These findings put the focus on the spliceosome as a potential target in anti-cancer treatment.

[0242] As already mentioned, few small molecules have already been reported to inhibit the spliceosome and specifically to inhibit the spliceosome factor SF3B1 function. Although the precise mechanisms of these small molecules are not yet completely understood, it has been reported that they can be effective in cancer therapy by reducing tumor size from 40 to 80% depending of the compound used. The only one to date that has been tested in human is the E7107. It has been stopped because of problems of toxicity. This compound is known to inhibit the spliceosome by interacting with SF3B1.

[0243] Moreover, Cytotoxic T lymphocytes failure to reject tumors can in part be explained by an initial inappropriate CTL activation by pAPCs. A defined subset of dendritic cells (DCs) has been described in the tumor microenvironment (TME) to be able to migrate to the tumor draining lymph nodes, deliver intact antigens encountered in the TME and prime directly or not naïve CD8.sup.+ T cells. In addition, it was suggested that some DCs are able to directly prime naïve CD8 T cells in the TME. Inventors recently demonstrated that tumor-associated PTPs are a source material for CD8.sup.+ T cells cross-priming by DCs and may mainly be transferred from tumor cells to DCs by PTPs-carrying exosomes. Besides, they provided hints that PTPs for endogenous and cross-presentation are produced by the same translation event and that the two pathways then diverge quickly. PTPs are rare products and the efficiency of PTPs vaccines or exosome-containing PTPs vaccines was shown to rely on the previous PTPs enrichment of PTPs proteasome inhibitor. Inventors believe that the splicing inhibitor Madrasin and its different derivatives, in addition to provide more source materials for the direct antigen presentation, enriches the pool of SL8-containing PTPs that serve as a source material for intratumoral DC uptake and cross-presentation, inducing an enhanced SL8-specific CD8.sup.+ T cell proliferation.

[0244] In the present description, inventors provide both in vitro and in vivo evidences that by modulating the spliceosome activity using specific derivative compounds from Madrasin, it is possible to induce a specific anti-tumor immune response against different cancer models. Madrasin has been reported to interfere in the early step of assembly of the spliceosome. In fact, it has been demonstrated that Madrasin inhibits the A complex of the pre-spliceosome to form a larger pre-catalytic spliceosome B complex. We demonstrated that by inhibiting the formation of the spliceosome, as early as possible using derivatives of the Madrasin in vitro and in vivo, the anti-tumor antigenic presentation was increased significantly, by inducing specifically CD8.sup.+ T cell proliferations against PTPs-dependent epitopes. They report that these derivatives can be used as chemotherapeutic agents against melanoma and sarcoma.

REFERENCES

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