Patent Application
20220296590
The present patent application refers, in a first aspect, to compounds which are phosphodiesterase enzyme inhibitors (PDE).
The present application refers, in a second aspect, to antiproliferative and antitumor compounds.
Said compounds can be used, for example, as anti-inflammatory, vasodilators, stimulants and inotropic in cardiac insufficiency and pulmonary diseases. Notably, the PDE inhibitors have showed themselves to be useful in the treatment of erectile dysfunction in men. Additionally, said compounds can be used as antiproliferative and antitumor.
The phosphodiesterase inhibitor compounds are the main agents used in medical clinic for the treatment and/or prevention of erectile dysfunction, disorders and/or conditions that are treatable with tissue relaxation and other diseases that are treatable with phosphodiesterase inhibitors. In the case of erectile dysfunction, the PDE-5 inhibitors are the ones most used in medical clinic.
The erectile dysfunction, more commonly known as sexual impotence is one of the diseases which is most harmful to the quality of life of a man. For a long time, the erectile dysfunction haunted men without much possibility of effective treatment.
Before the 70s, nearly all the erectile dysfunction cases were considered as resulting from psychological causes, and the treatments consisted in the empirical administration of testosterone or referral to a psychiatrist.
The oral treatment arose from clinical research on the use of cGMP-PDE inhibitors, more specifically, the PDE-5 inhibitors. The precursor of these compounds was the 5-[2-ethoxy-5-(4-methylpiperazinyl -sulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H -pyrazole[4,3-d]pyrimidine-7-one, or sildenafil, with vasodilator properties and which potentializes the effects of the nitric oxide. The sildenafil molecule was originally described in North-American patent U.S. Pat. No. 5,250,534.
Subsequently, other inhibitor compounds of the PDE-5 enzyme were developed and are cited in a large number of technical literature publications, as well as in patent publications. Among the known compounds we highlight: the vardenafil molecule, originally described in patent U.S. Pat. No. 3,635,178; the tadalafil, originally described in American patent U.S. Pat. No. 5,859,006; The BL-106, BL-230 and BL-236 compounds which are not commercialized yet, originally described in American patents U.S. Pat. No. 8,338,432 and U.S. Pat. No. 9,359,378 and developed by the same research team as the present patent application.
Additionally, the researchers verified that the tissue relaxant compounds have been used to promote the relaxation of several tissues with the purpose of treating or acting as adjuvant in the treatment, procedure or surgery related to lithiasis (Korkes, F. et al, J Bras Nefrol. (2009) 31(1):55), prostate enlargement (for example, prostatic hyperplasia, prostatitis) (WO 9911279) and urethral constriction (Van der werf et al, BJU International (2002) 90:588). Other tissue relaxant treatable conditions and/or disorders are known and described in the state of the art.
In view of this scenario and seeking new alternatives of phosphodiesterase inhibitor compounds, more specifically as PDE-5 inhibitors, for the treatment of erectile dysfunction, tissue relaxant treatable conditions and/or disorders and other diseases that are treatable with phosphodiesterase inhibitors, the researchers of the present invention developed new compounds which are now described and claimed, comprising the compound 3-(benzo[d][1,3]dioxol-5-yl)-7-(1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione and the stereoisomers thereof which presented, together and separately, prominent activities as PDE inhibitors, antiproliferative and antitumor.
In this manner, under one aspect, the compounds now claimed are presented as alternatives for the treatment of several diseases and medical conditions which benefit from the PDE inhibition, such as erectile dysfunction, cardiac insufficiency, pulmonary hypertension and for the treatment of several medical diseases and conditions which benefit from the antiproliferative and antitumor effects.
The present patent application refers, in one aspect to the compound 3-(benzo[d][1,3]dioxol-5-yl)-7-(1-hydroxypropan -2-yl)-1-(1h-indole-3-yl)-6,7-dihydro-3h-oxazole[3,4-a]pyrazine-5,8-dione of formula (I) and the stereoisomers thereof,
as well as their salts, solvates, hydrates, prodrugs and pharmaceutically acceptable esters;
In alternative aspects, the referred stereoisomers of the compound of formula (I) are in their individual separate forms and/or in the forms of racemic mixtures or non-racemic mixtures with diastereoisomeric excess in any proportions.
In specific aspects, the stereoisomers are the compounds of formulas (II) and (III)
The compound of formula (II) is named 3-(benzo[d][1,3]dioxol-5-yl)-7-((S)1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-236) and the compound of formula (III) is named 3-(benzo [d][1,3]dioxol-5-yl)-7-((R)1-hydroxypropan-2-yl)-1-(1H- indole-3-yl)- 6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-239).
In a preferred aspect, the present patent application refers to the isolated stereoisomers known by the formulas (IV), (V), (VI) and (VII), below:
The stereoisomers described above are named:
Compound of formula (IV): (S)-3-(benzo[d][1,3]dioxol-5- yl)-7-((S)-1-hydroxypropan-2-yl)-1-(1H -indole-3-yl)-6,7- dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-236A);
Compound of formula (V): (R)-3-(benzo [d][1,3]dioxol-5-yl)-7-((S)-1-hydroxypropan-2-yl)-1-(1H -indole-3-yl)-6,7- dihydro-3H-oxazole[3,4-a] pyrazine-5,8-dione (BL-236B);
Compound of formula (VI): (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan-2-yl)-1-(1H -indole-3-yl-6,7- dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-239A); and
Compound of formula (VII): (R)-3-(benzo[d][1,3]dioxol-5- yl)-7-((R)-1-hydroxypropan-2-yl)-1-(1H -indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-239B).
Another aspect of the present patent application refers to mixtures of stereoisomers of the compounds 3-(benzo[d][1,3]dioxol-5-yl)-7-(1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)- 6,7-dihydro-3H -oxazole[3,4-a]pyrazine-5,8-dione of formula (I), wherein the mixtures can be a racemic mixture or a non-racemic mixture.
Preferably, the present patent application seeks protection for non-racemic mixtures with diastereoisomeric excess in any proportions.
More specifically, the diastereoisomeric proportion is in the range from 1:99 to 99:1. In another preferred aspect, the mixture is in the range from 1:50 to 50:1, alternatively in the range from 1:25 to 25:1. In a more preferred aspect, the non-racemic mixtures with diastereoisomeric excess are in the range from 1:2 to 2:1, alternatively in the proportion of 1:1.
In another preferred aspect, the present patent application refers to a mixture of the diastereoisomerics described in formulas (VIII) and (IX), whereby the mixture can contain any proportions of the diastereoisomerics.
The mixture of diastereoisomerics of formula (VIII) is named (R)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R) -1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione with (S)-3- (benzo [d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan -2-yl)-1-(1H-indole-3-yl)-6, 7-dihydro-3H-oxazole[3, 4-a]pyrazine-5,8-dione (BL-380) and presents the following structure:
Wherein the diastereoisomeric proportion is in the range from 1:99 to 99:1. In another preferred aspect, the mixture is in the range from 1:50 to 50:1, more preferably, in the range from 1:25 to 25:1. In a more preferred aspect, the mixture is in the range from 1:2 to 2:1, more preferably in the 1:1 proportion.
The mixture of diastereoisomerics of formula (IX) is named (R)-3-(benzo[d][1,3]dioxol-5-yl)-7-(S)-1-hydroxypropan-2-yl)-1- (1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione with (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((S)-1-hydroxypropan-2-yl) -1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-241), and presents the following structure:
wherein the diastereoisomeric proportion is in the range from 1:99 to 99:1. In another preferred aspect, the mixture is in the range from 1:50 to 50:1, alternatively in the range from 1:25 to 25:1. In a more preferred aspect, the mixture is in the range from 1:2 to 2:1, alternatively in the 1:1 proportion.
The diastereoisomerics and mixtures thereof, which are the object of the present patent application, can be prepared, for example, according to the following reaction schemes:
The present patent application further covers the pharmaceutical compositions comprising the referred stereoisomers of the compound of formula (I) in their individual separate forms and/or in the forms of racemic mixtures or non-racemic mixtures with stereoisomer excess in any proportions and drugs comprising the referred stereoisomers. Said compositions can be prepared according to methods that are known in the pharmaceutical technology, using thinners, excipients or pharmaceutically acceptable carriers.
Another aspect of the present patent application refers to the use of the compound of formula (I) and the stereoisomers thereof such as antitumor and phosphodiesterase enzyme inhibitors of the type 5 (PDE-5); and to the use of the referred stereoisomers in the treatment of benign prostatic hyperplasia and cancer, more specifically, prostate cancer.
In another preferred aspect the present patent application refers to the use of a mixture defined by the formula (VIII) and named as (R)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan-2-yl)-1-(1H-indole -3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione with (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H -oxazole[3,4-a]pyrazine-5,8-dione (BL-380), in any proportion of the diastereoisomers, as antitumor agent and for the treatment of disorders that are treatable with PDE-5 inhibitors in mammals. Particularly for the treatment of prostate cancer and benign prostatic hyperplasia.
Another preferred aspect of the present patent application refers to the use of isolated stereoisomers defined by the formulas (IV), (V), (VI) and (VII) and named, respectively, as: (S)-3- (benzo[d][1,3]dioxol-5-yl)-7-((S)-1-hydroxypropan-2-yl)-1-(1H -indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-236A) ; (R)-3-(benzo[d][1,3]dioxol-5-yl) -7-((S)-1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-236B); (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R) -1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H -oxazole[3,4-a]pyrazine-5,8-dione (BL-239A) ; and (R) -3- (benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan -2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-239B), as antitumor agent and for the treatment of disorders that are treatable with PDE-5 inhibitors in mammals. In particular for the treatment of prostate cancer and benign prostatic hyperplasia.
Additional objects of the present patent application are the uses of the compound of formula (I) and the stereoisomers thereof, salts, esters, prodrugs, as herein described, in the preparation of drugs antiproliferative, antitumor and phosphodiesterase enzyme inhibitors, more specifically inhibitors of the type 5 phosphodiesterase enzyme (PDE-5); and to the use of the referred stereoisomers in the preparation of medicament for the treatment of diseases and disorders, such as inflammatory diseases and disorders, cardiovascular, pulmonary, erectile dysfunction in man, benign prostatic hyperplasia and cancer, more specifically, prostate cancer.
The present patent application further describes a method for treating cancer, such as benign prostatic hyperplasia and cancer, more specifically prostate cancer, using the stereoisomers of the formula (I), the salts, hydrates, prodrugs thereof and pharmaceutically acceptable esters; wherein the stereoisomers are in their individual separate forms of racemic mixtures or non-racemic mixtures with diastereoisomer excess in any proportions.
Said uses and treatment methods described above comprise the supply of a therapeutically effective amount of the compounds and their respective salts, solvates, prodrugs, stereoisomers and racemic mixtures of the same.
Another object of the present patent application is the process for the production of stereoisomers of the compound 3-(benzo[d][1,3]dioxol-5-yl)-7-(1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione of formula (I), as described in the above schemes and the following examples.
It must be understood that the examples and embodiments described in detail herein illustrate the present patent application without, however, limiting the scope thereof.
In a 500 mL balloon with mechanical stirrer, addition funnel and drying tube containing CaCl2, there were added the intermediary compound (E)-methyl 2-((benzo[d][1,3]dioxol-5-yl methylene)amine)-3-(1H-indole-3-yl) -3-oxopropanoate of formula (A) (5.0 g, 13.7 mmol), dry THF (90 mL) and dry pyridine (5.5 mL) which was stirred for 30 minutes. To this suspension was added, slowly, approximately 3 h, a solution of chloroacetyl chloride (1.5 mL, 19.2 mmol) in dry THF (23 mL). After the addition, the reaction mixture was reacted for a period of 4 h at room temperature. At the end of this period, (R)-2-aminopropane-1-ol (23.7 g, 315.1 mmol) was added to the medium, and the mixture was stirred for a period of 16 h. At the end of this time, 100 mL ethanol was added. The system was submitted to a vacuum distillation, and the excess solvent removed up to the precipitation of the product. The balloon was cooled to room temperature and subsequently between 0 and 5° C., and stirred for 2 h at this temperature. The solid was placed in a 100 mL balloon and 30 mL of ethanol was added; the mixture was stirred for a period of 30 minutes. Upon conclusion of this period, the solid was filtered and vacuum dried, leading to the forming of the compound of formula (VIII), constituted by a mixture of the compounds (VI) and (VII) corresponding to 41% yield.
The compound obtained by this method presents IUPAC nomenclature (R)-3-(benzo[d][1,3]dioxol-5-yl) -7-((R)-1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione with (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-380), represented by the chemical structure (VIII):
and presents the following characteristics: yellow solid; fusion point 256-262° C.; NMR H (500 MHz, DMSO-d6): δ 11.84 (1s, 2H), 8.97-8.95 (m, 2H), 7.87-7.85 (m, 2H), 7.47-7.46 (m, 2H), 7.17-7.09 (m, 8H), 7.08-7.04 (m, 2H), 6.04 (s, 4H), 4.84 (t, J=5.8 Hz, 1H), 4.80 (t, J=5.8 Hz, 1H), 4.67-4.60 (m, 2H), 4.14-4.03 (m, 4H), 3.57-3.42 (m, 4H), 1.11 (d, J=7.0 Hz, 3H), 1.08 (d, J=7.0 Hz, 3H); NMR 13C (125 MHz, DMSO-d6): δ 159.0, 158.7, 157.0, 156.7, 148.3, 147.6, 147.57, 147.55, 135.77, 135.76, 131.1 131.0, 130.9, 130.8, 125.2, 122.1, 121.3, 120.50, 120.49, 120.44, 120.36, 112.1, 108.3, 108.2, 106.5, 106.4, 105.2, 105.1, 102.3, 101.4, 90.3, 90.1, 61.8, 61.4, 49.4, 49.3, 46.3, 46.1, 13.2, 13.1. HRMS (EI) m/z calculated for C24H21N3O6: [447.1430]+found: 448.1542 [M+H]+.
The following compounds were used as intermediary reactions: (E)-methyl 2-((benzo[d][1,3]dioxol-5-yl methylene)amine)-3-(1H-indole-3-yl)-3-oxopropanoate, described by the chemical structure (A); the compound (R)-2-aminopropane-1-ol (3), and the 2-chloroacetyl chloride (1), as exemplified below and in scheme 1.
In a 500 mL balloon with mechanical stirrer, addition funnel and drying tube containing CaCl2, there were added the intermediary compound (E)-methyl 2-((benzo[d][1,3]dioxol-5-yl methylene)amine)-3-(1H-indole-3-yl) -3-oxopropanoate of formula (A) (5.0 g, 13.7 mmol), dry THF (90 mL) and dry pyridine (5.5 mL) which was stirred for 30 minutes. To this suspension was added, slowly, approximately 3 h, a solution of chloroacetyl chloride (1.5 mL, 19.2 mmol) in dry THF (23 mL). After the addition, the reaction mixture was reacted for a period of 4 h at room temperature. At the end of this period, (R)-2-aminopropane-1-ol (23.7 g, 315.1 mmol) was added to the medium, and the mixture was stirred for a period of 16 h. At the end of this time, 100 mL ethanol was added. The system was submitted to a vacuum distillation, and the excess solvent removed up to the precipitation of the product. The balloon was cooled to room temperature and subsequently between 0 and 5° C., and stirred for 2 h at this temperature. The solid was placed in a 100 mL balloon and 30 mL of ethanol was added; the mixture was stirred for a period of 30 minutes. Upon conclusion of this period, the solid was filtered and vacuum dried, leading to the forming of the compound of formula (IX), constituted by a mixture of the compounds (IV) and (V) corresponding to 40% yield.
The compound obtained by this process presents IUPAC nomenclature (R)-3-(benzo[d][1,3]dioxol-5-yl)-7-((S)-1-hydroxypropan-2-yl)-1-(1H-indole -3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione with (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((S)-1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H -oxazole[3,4-a]pyrazine-5,8-dione (BL-241), represented by the chemical structure (IX):
and presents the following characteristics: yellow solid; fusion point 256-261° C.; NMR 1H (500 MHz, DMSO-d6): δ11.84 (is, 2H), 8.97-8.95 (m, 2H), 7.88-7.85 (m, 2H), 7.47-7.46 (m, 2H), 7.17-7.05 (m, 10H), 6.98-6.96 (m, 2H), 6.04 (s, 4H), 4.84 (t, J=5.5 Hz, 1H), 4.80 (t, J=5.5 Hz, 1H), 4.67-4.60 (m, 2H), 4.14-4.03 (m, 4H), 3.57-3.42 (m, 4H), 1.11 (d, J=7.0 Hz, 3H), 1.08 (d, J=7.0 Hz, 3H); NMR 13C (125 MHz, DMSO-d6) : δ 159.0, 158.7, 157.0, 156.8, 148.3, 147.6, 147.57, 147.55, 135.8, 135.78, 135.77, 131.1 131.0, 130.9, 130.8, 125.2, 122.1, 121.3, 120.5, 120.49, 120.44, 120.36, 112.1, 108.3, 108.2, 106.5, 106.4, 105.2, 105.1, 102.3, 101.4, 90.3, 90.1, 61.8, 61.4, 49.4, 49.3, 46.3, 46.1, 13.2, 13.1. HRMS (EI) m/z calculated for C24H21N3O6: [447.1430]+found: 448.1541 [M+H]+.
The following compounds were used as intermediary reactions: (E)-methyl 2- ((benzo[d][1,3]dioxol-5-yl methylene)amine)-3-(1H-indole-3-yl)-3-oxopropanoate, described by the chemical structure (A); and the compound (S)-2-aminopropane-1-ol (4) and the chloroacetyl chloride (1) as exemplified below and in scheme 2.
In a 1.0 L balloon there were added 4.5 g of the compound of formula (IX) (R)-3-(benzo[d][1,3]dioxol-5-yl)-7-((S)-1- hydroxypropan-2-yl)-1-(1H -indole-3-yl)-6,7-dihydro-3H- oxazole[3,4-a]pyrazine-5,8-dione with (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((S) -1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro -3H-oxazole[3,4-a]pyrazine-5,8- dione (BL-241), and 75 mL of a mixture of THF/H2O (8:2). The suspension was heated at 50° C. up to the solubilization. After the solubilization, 560 mL of methanol was added to the medium and stirred for 2 hours at room temperature. The solid formed was vacuum filtered, washed in 50 mL of methanol, and kiln dried at 100° C. for 4 hours. This step generated the isolation of one of the compounds BL-236A or BL-236B, which presents the following characteristics: yellow solid, fusion point: 227-231° C.; NMR 1H (300 MHz , DMSO-d6): δ 11.89 (1s, 1H), 8.97 (d, J=1.8 Hz, 1H), 7.86 (d, J=7.9 Hz, 1H), 7.46 (d, J=7.9 Hz, 1H), 7.18-7.03 (m, 5H), 6.98 (dd, J=7.4 Hz, J=1.0 Hz, 1H), 6.04 (s, 2H), 4.88 (t, J=5.5 Hz, 1H), 4.69-4.58 (m, 1H), 4.07 (dd, J=22.5 Hz, J=17.5 Hz, 2H), 3.58-3.41 (m, 2H), 1.07 (d, J=6.9 Hz, 3H) ; NMR 13C (75 MHz, DMSO-d6) : δ 159.9, 157.1, 148.4, 147.7, 147.6, 135.8, 131.2, 130.9, 125.3, 122.2, 121.4, 120.6, 120.5, 112.3, 108.4, 106.6, 105.2, 102.5, 101.5, 90.2, 61.4, 49.4, 46.1, 13.2; HRMS (EI) m/z calculated for C24H21N3O6: [447.1430]+, found: 448.1321 [M+H]+. The chemical structure of the compound was elucidated by nuclear magnetic resonance analysis, high resolution mass and X-ray crystallography.
The mother liquor of the previous filtration was returned to the reaction balloon and 200 mL of H2O were slowly added, leading to turbidity of the reaction medium, which was stirred for one hour at room temperature. The suspension was vacuum filtered and washed with 50 mL ethanol. The solid obtained was resuspended in 50 mL of ethanol and filtered once more, which was subsequently kiln dried at 100° C. for 4 hours, leading to the isolation of the other compound of BL-236), which presents the following characteristics: yellow solid, fusion point: 221-225° C.; NMR 1H (300 MHz , DMSO-d6): δ 11.90 (1s, 1H), 8.96 (s, 1H), 7.85 (d, J=7.9 Hz, 1H), 7.46 (d, J=7.9 Hz, 1H), 7.18-7.03 (m, 5H), 6.97 (d, J=7.9 Hz, 1H), 6.04 (s, 2H), 4.83 (t, J=5.7 Hz, 1H), 4.67-4.60 (m, 1H), 4.09 (dd, J=25.7 Hz, J=17.6 Hz, 2H), 3.56-3.41 (m, 2H), 1.10 (d, J=6.9 Hz, 3H) ; NMR 13C (75 MHz, DMSO-d6): δ 158.7, 156.8, 148.4, 147.6 (2C), 135.8, 131.1, 130.9, 125.3, 122.2, 121.4, 120.6, 120.5, 112.2, 108.3, 106.6, 105.2, 102.4, 101.5, 90.4, 61.8, 49.4, 46.3, 13.3; HRMS (EI) m/z calculated for C24H21N3O6: [447.1430]+, found: 448.1530 [M+H]+. The chemical structure of the compound was elucidated by nuclear magnetic resonance analysis, high resolution mass and X-ray crystallography.
With this process we have the separation of the compounds (S) 3-(benzo[d][1,3]dioxol-5-yl)-7-((S) -1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro- 3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-236A) and (R) -3- (benzo[d][1,3]dioxol-5-yl)-7-((57)-1-hydroxypropan -2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-236B), represented by the structures (IV) and (V), respectively.
In a 1.0 L balloon there were added 4.5 g of the compound of formula (VIII) (R)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan-2-yl)-1-(1H -indole-3-yl)-6,7-dihydro-3H- oxazole[3,4-a]pyrazine-5,8-dione with (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R) -1-hydroxypropan-2-yl)-1-(1H- indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8- dione (BL-380), and 75 mL of a mixture of THF/H2O (8:2). The suspension was heated at 50° C. up to the solubilization. After the solubilization, 560 mL of methanol was added to the medium and stirred for 2 hours at room temperature. The solid formed was vacuum filtered, washed in 50 mL of methanol, and kiln dried at 100° C. for 4 hours. This step generated the isolation of one of the compounds BL-236A or BL-236B, which presents the following characteristics: yellow solid, fusion point: 233-236° C. ; NMR 1H (300 MHz , DMSO-d6): δ 11.89 (1s, 1H), 8.97 (d, J=2.4 Hz, 1H), 7.86 (d, J=7.9 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H), 7.18-7.04 (m, 5H), 6.98 (d, J=8.3 Hz, 1H), 6.04 (s, 2H), 4.88 (t, J=5.7 Hz, 1H), 4.67-4.60 (m, 1H), 4.07 (dd, J=22.5 Hz, J=17.5 Hz, 2H), 3.58-3.41 (m, 2H), 1.06 (d, J=6.9 Hz, 3H); NMR 13C (75 MHz, DMSO-d6): δ 159.1, 157.1, 148.4, 147.7, 147.6, 135.8, 131.2, 130.9, 125.3, 122.2, 121.4, 120.6, 120.5, 112.3, 108.4, 106.6, 105.2, 102.4, 101.5, 90.2, 61.4, 49.4, 46.1, 13.2; HRMS (EI) m/z calculated for C24H21N3O6: [447.1430]+, found: 448.1540 [M+H]+. The chemical structure of the compound was elucidated by nuclear magnetic resonance analysis, high resolution mass and X-ray crystallography
The mother liquor of the previous filtration was returned to the reaction balloon and 200 mL of H2O were added slowly, leading to turbidity of the reaction medium, which was stirred for one hour at room temperature. The suspension was vacuum filtered and washed with 50 mL ethanol. The solid obtained was resuspended in 50 mL of ethanol and filtered once more, which was subsequently kiln dried at 100° C. for 4 hours, leading to the product of interest (another BL-239 molecule) Yellow solid, fusion point: 223-225° C. ; NMR 1H (300 MHz , DMSO-d6): δ 11.90 (1s, 1H), 8.96 (s, 1H), 7.85 (d, J=8.2 Hz, 1H), 7.47 (d, J=8.2 Hz, 1H), 7.18-7.03 (m, 5H), 6.98 (d, J=8.0 Hz, 1H), 6.04 (s, 2H), 4.84 (t, J=5.6 Hz, 1H), 4.69-4.58 (m, 1H), 4.09 (dd, J=25.7 Hz, J=17.6 Hz, 2H), 3.55-3.41 (m, 2H), 1.10 (d, J=6.7 Hz, 3H) ; NMR 13C (75 MHz, DMSO-d6): δ 158.7, 156.8, 148.4, 147.6 (2C), 135.8, 131.1, 130.9, 125.3, 122.2, 121.4, 120.6, 120.5, 112.2, 108.3, 106.6, 105.2, 102.4, 101.5, 90.4, 61.8, 49.4, 46.3, 13.3; HRMS (EI) m/z calculated for C24H21N3O6: [447.1430]+, found: 448.1529, [M+H]+. The chemical structure of the compound was elucidated by nuclear magnetic resonance analysis, high resolution mass and X-ray crystallography.
With this process we have the isolation of the compounds (S)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan-2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H -oxazole[3,4-a]pyrazine-5,8-dione (BL-239A) and (R)-3-(benzo[d][1,3]dioxol-5-yl)-7-((R)-1-hydroxypropan -2-yl)-1-(1H-indole-3-yl)-6,7-dihydro-3H-oxazole[3,4-a]pyrazine-5,8-dione (BL-239B), represented by the structures (VI) and (VII) respectively.
The evaluation of the compound is studied using BPS Bioscience enzyme assay kit (catalogue 60350). The PDE5A1 Assay kit is designed for identification of PDE5A1 inhibitors using fluorescence polarization. The assay is based on the binding of a fluorescent nucleotide monophosphate generated by PDE5A1 enzyme to the binding agent.
In this methodology only two simple steps on a microtiter plate are required for PDE5A1 reactions. First, the fluorescently labeled cGMP is incubated with PDE5A1 for 1 hour. Second, the binding agent is added to the reaction mixture to produce a change in fluorescent polarization that can then be measured using a fluorescence reader equipped for the measurement of fluorescence polarization.
For the assay the storage reagent FAM-Cyclic-3′, 5′-GMP (20 μM) is diluted with PDE assay buffer to achieve the solution with a 200 μM concentration; the PDE5A1 enzyme is thawed and diluted with the PDE buffer obtaining a PDE5A1 solution at 10 pg/mL.
The assay compounds are diluted with DMSO 100% (v/v) at a concentration of 1000 μM and subsequently diluted in a Sodium chloride 0.9% solution to obtain a curve of concentrations of interest for the assay. The compounds are diluted in concentrations of: 10 nM, 30 nM, 50 nM, 100 nM, 300 nM, 500 nM, 1 μM, 3 μM, 5 μM (DMSO 10% v/v).
The study is comprised of groups: (i) substrate control; (ii) positive control (KIT); (iii) samples in several concentrations; (iv) tadalafil (comparative control). Each parameter (group) is triple labeled.
For the 4 groups there is added, to each microplate well, 25 mL of the FAM-Cyclic-3′ reagent, 5′-GMP (200 μM).
To the substrate control group there is added, to each well, 5 mL of vehicle (DMSO 10% v/v) and 20 mL PDE Buffer.
To the positive control group there is added, to each well, 5 mL of vehicle (DMSO 10% v/v).
To the tadalafil group: there is added 5 mL of the solution in the concentrations of interest.
To the test group there is added 5 mL of the sample solution (test compound)
OBSERVATION: the parameters tested are in triplicate.
To begin the reaction, add 20 mL of the PDE5A1 enzyme (diluted as described above) to the microplate wells designated as Positive Control and Samples (test and tadalafil), and incubate the microplate, for 1 hour, at a temperature of 25° C.
After the incubation period, the reaction is interrupted by the addition, in all the wells, of 100 mL of the binding agent solution and the microplate is incubated for 30 minutes, under light stirring, at room temperature. The reading of the microplate is carried out in Polarized Fluorescence (475-495 nm—excitation and 518-538 nm—emission detection) 500 nanoseconds integration time.
For the compound BL-380 there were prepared the concentrations of 10 nM, 30 nM, 50 nM, 100 nM, 300 nM, 500 nM, 1 μM (1000 nM), 3 μM (3000 nM) and 5 μM (5000 nM). Based on the results obtained, (table 1) it is possible to observe that the compound BL-380 was capable of inhibiting the PDE5 in a dose-dependent manner, however, this inhibition was only observed as from the concentration of 300 nM of BL-380.
For the compound BL-241 there were prepared concentrations of 500 nM, 1 μM (1000 nM) and 3 μM (3000 nM). Based on the results obtained (table 2) it is possible to observe that the compound BL-241 was also capable of inhibiting the PDE5 in a dose-dependent manner.
For the execution of the assay, the compounds are diluted in DMSO 100% (v/v) at a concentration of 1000 μM and subsequently diluted in a Sodium Chloride solution 0.9% to obtain the storage solutions of interest for use in the assays. The final concentration of interest will be directly diluted in the cell culture medium.
For the assay there are used the following tumor cell lineages: PC3-human prostate cancer cells; LNCAP-human prostate cancer cells; DU145-human prostate cancer; MNK45-human gastric cancer and RT4-human bladder cancer cells.
The tumor cell lineages are maintained in culture according to standard procedures established by the Cell Bank of Rio de Janeiro (lineage place of origin).
For the assay, the tumor cells are seeded in the concentration of 10.000 cells per well (final volume of the reaction of 200. mL), the plate is incubated for 8 hours, for the cells to adhere to the wall of the wells. After this period, the plate is divided in groups:
OBSERVATION: the treatment with the concentrations of samples (test compounds) and of the controls are carried out in triplicate.
Incubate the plate for 72 hours in a CO2 kiln at 37° C.
After the incubation period, add 100 mL/well of the solution with DNA marker from the CYQuant Kit and incubate the plate, for 60 minutes, in a CO2 incubator at 37° C. The fluorescence intensity is determined using a fluorescence microplate reader with 485 nm excitation and 530 nm emission detection. The results obtained were normalized relative to the positive control group (100% proliferation).
For the compounds there are prepared the storage solutions in the concentrations of: 100 nM, 1 μM, 3 μM, 5 μM, 10 μM and 30 μM (DMSO 10% v/v). While the final concentrations in the test are of: 10 nM, 100 nM, 300 nM, 500 nM, 1000 nM and 3000 nM (in DMSO 1% v/v).
The results obtained demonstrate that the BL-380 compound presented antiproliferative effect for the 3 prostate cancer tumor lineages tested PC3, LNCAP and DU145) and for the bladder cancer tumor lineage (RT4), as can be observed in table 3. And the BL-241 compounds presented antiproliferative effect for the 3 prostate cancer tumor lineages tested (PC3, LNCAP and DU145) and for the bladder cancer tumor lineage (RT4), as can be observed in table 4.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/BR2020/050259 | 7/14/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62874321 | Jul 2019 | US |
