Title of Invention	DERIVATIVES OF ISOSORBIDE MONONITRATE AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME FOR TREATMENT OF CARDIOVASCULAR OR CORONARY DYSFUNCTIONS, OR FOR TREATMENT OF DYSFUNCTIONS OF CIRCULATORY SYSTEM.
Abstract	Novel derivatives of isosorbide mononitrate and its pharmaceutically acceptable salts, which have vasodi- 5 lating activity with a reduced effect of tolerance, of the general formula (I) in which A and B individually represent any of the groups 10 -ONO2 and -Z-CO-R, wherein Z is an oxygen atom or sul- phur atom and R is an alkyl C1 - C4 group, an aryl group or an aralkyl group, eventually substituted, or the group 15 in which R1 is hydrogen, or an alkyl C1 - C4 group, an aryl group or an aralkyl group, eventually substituted, with the proviso that one of A or B is always -ONO2, but never both of them at the same time, when Z is an sul- phur atom R is an alkyl C1 - C4 group, an aryl group or an 20 aralkyl group, eventually substituted, and when Z is an oxygen atom R is the group

Title of Invention

DERIVATIVES OF ISOSORBIDE MONONITRATE AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME FOR TREATMENT OF CARDIOVASCULAR OR CORONARY DYSFUNCTIONS, OR FOR TREATMENT OF DYSFUNCTIONS OF CIRCULATORY SYSTEM.

Abstract

Novel derivatives of isosorbide mononitrate and its pharmaceutically acceptable salts, which have vasodi- 5 lating activity with a reduced effect of tolerance, of the general formula (I) in which A and B individually represent any of the groups 10 -ONO2 and -Z-CO-R, wherein Z is an oxygen atom or sul- phur atom and R is an alkyl C1 - C4 group, an aryl group or an aralkyl group, eventually substituted, or the group 15 in which R1 is hydrogen, or an alkyl C1 - C4 group, an aryl group or an aralkyl group, eventually substituted, with the proviso that one of A or B is always -ONO2, but never both of them at the same time, when Z is an sul- phur atom R is an alkyl C1 - C4 group, an aryl group or an 20 aralkyl group, eventually substituted, and when Z is an oxygen atom R is the group

Full Text	DERIVATIVES OF ISOSORBIDE MONONITRATE AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME FOR TREATMENT OF CARDIOVASCULAR OR CORONARY DYSFUNCTIONS, OR FOR TREATMENT OF DYSFUNCTIONS OF CIRCULATORY SYSTEM. Field of the invention The present invention relates to novel derivatives of isosorbide mononitrate which have a potent vasodilating activity and which, at the same time, have a significantly reduced tolerance, and in particular, pharmaceutical composition containing the same for treatment of cardiovascular or coronary dysfunctions, or for treatment of dysfunctions of circulatory system. Background art The nitric acid esters of organic: compounds, common known as nitrated organic compounds, are known and have been used as vasodilating agents. Within these, the usefulness of mono and di-nitrated isosor- bide is well known, and further there have been de- scribed compounds with vascular and coronary activities based on substitution reactions of the free hydroxyl group of isosorbide mononitrate- For example, the US-A- 4891373 patent describes derivatives of aminepropanol corresponding to the formulas for the treatment of the angina pectoris and systemic and pulmonary hypertension. The US-A-5665766 patent describes the isosor- bide 5-mononitrate 2-acetylsalicylate, of formula as well as its platelets anti-aggregating activity. One of the principal problems of the nitrated organic compounds mentioned above resides on the fact that these are quite sensible in relation to the phe- nomena known as tachyphylaxy or tolerance, which re- lates to that its effect on the organism decreases dur- ing prolonged treatment, and it is then required to sensitively elevate the administrated doses in a gradu- ated manner or otherwise to perform a pharmacologically wash out. It is also known that one way of reducing the tolerance of the nitrated organic compounds consists of introducing a thiol group in the molecule, for example by use of sulphur containing amino acids. The European patent EP-B-0362575 describes nitrated organic com- pounds with incorporated Cysteine and/ mainly, Methion- ine molecules. The patent application WO-A-92/04337 de- scribes organic nitrated derivatives of the ring of the thiazolidine with vasodilating activity and a reduced tolerance. The patent application WO-A-93/03037 de- scribes an enormously amount of different nitrated or- ganic vasodilating compounds, with reduced tolerance, of highly variable structures. Within these are in- cluded generically, i.e. without specifying nor de- scribing one single specific product, derivatives of isosorbide mononitrate according to following structure in which R5 represents a hydrogen atom, a C1-C6 alkyl group, a phenyl, etc. The nitrated organic compounds described in the documents mentioned above do not in itself solve the problems originating from the tolerance of the ni- trated organic compounds, since these still have prob- lems in relation to low vasodilating activity, high tolerance, etc.. Accordingly, it is still necessary to develop novel nitrated organic compounds which have a high vasodilating activity combined with a more de- creased level of tolerance being maintained persis- tently. Summary of the invention An object of the invention is a novel type of compounds, derivatives of isosorbide mononitrate, which are capable of providing a potent vasodilating effect and which at the same time show a small or null toler- ance effect. A further object of the present invention re- lates to the use of the novel derivatives of isosorbide mononitrate for the manufacture of a medicament for the treatment of disorders related to dysfunctions of the circulatory system, in particular at the level of the coronary system. Detailed description of the invention According to the present invention there is provided a derivative of isosorbide mononitrate, or a pharmaceutically acceptable salt thereof, having the following general formula (I) wherein one of A and B represents -ONO2 and the other represents wherein: either (i) Z is a sulphur atom and R is an optionally substituted C1-C4 alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group; or (ii) Z is an oxygen atom and R is the group v/herein R1 is hydrogen or an optionally substituted C1-C4 alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group. Within the novel derivatives of the invention it is preferred that when Z is a sulphur atom, R is. a short chain C1-C4 alkyl group, and when Z is an oxygen atom, R1 is a hydrogen atom or a short chain C1-C4 alkyl group. More preferably, within above mentioned crite- ria, B is the -ONO2 group, i.e. the compounds wherein the nitrate ester is at position 5 in the ring-shaped system of the isosorbide. The preferences mentioned above should not in any way be considered as limiting the scope of the ob- ject of the present invention. In case R1 is hydrogen the compounds of the invention could be represented as any of its two tautomers and both of the tautomer structures should be consid- ered as within the object of the invention. Examples of specific compounds within the ob- ject of the invention.could be following: isosorbide 2- (2'-ethylthio)nicotinate 5-mononitrate, of formula isosorbide 5-(2'-ethylthio)nicotinate 2-mononitrate, of formula isosorbide 2-(2'-mercapto)nicotinate 5-mononitrate, of formula isosorbide 5-{2'-mercapto)nicotinate 2-mononitrate, of formula 2-acetylmercaptoisosorbide 5-mononitrate, of formula isosorbide 2-(2'-methylthio)nicotinate 5-mononitrate, of formula isosorbide 5-(2'-ethylthio)nicotinate 2-mononitrate, of formula as well as the pharmaceutically acceptable salt of these, in particular their chlorhydrates. The compounds 1 and its chlorhydrate and the compound 5 are particularly preferred. The compounds of the present invention can be obtained by techniques of esterification using known or accessible starting products described in the basic or- ganic chemical literature known to the skilled person, for example the publications of Chemical Abstracts Ser- vice, the Beilstein Encyclopedia of organic products, or in any other appropriate publication available at university libraries. For example, when Z is an oxygen atom the compounds may be obtained from isosorbide or the corre- sponding isosorbide mononitrate through a reaction of esterification of these with the corresponding carbox- ylic acid or an activated derivative of this, for exam- ple an acid chloride, an anhydride, an active ester, etc. If the starting product is isosorbide, it will be necessary finishing with a further step consisting of nitrating the free hydroxyl group of the isosorbide, a thing which is not necessary if there is started from any of the isosorbide mononitrates in position 5 or in position 2 of the ring-shaped structure of said com- pound . When R1 is hydrogen, these compounds have a thiol group which can be unintentionally oxidized producing disulphur dimers. In this case, the dimers can be reverted to the corresponding monomers by reac- tion with triphenylphosphine in water, as described in R. Humphrey (1964), Analytical Chem,36,1812 y L.E.Overman (1974), Synthesis, 59. When Z is a sulphur atom the situation is very similar since it is enough to start from the cor- responding thiocarboxylic acid, in stead of the carbox- ylic acid mentioned above, and to use techniques well known to the expert for the formation of the thioester bond. On the other hand, if any of the reactions imply the epimerization of a chiral center, there may be used as a starting compound the adequate enantiomer of the isosorbide, for example the isomanide. The tests performed demonstrate that the novel isosorbide mononitrate derivatives of the inven- tion show a vasodilating activity comparable, as a minimum, with that of the isosorbide mononitrate by it- self, and in some cases highly superior. Further, they manifest a significant inferior tolerance as compared to that observed with said compound and in some cases it approaches practically null. Consequently, the compounds of the invention may very efficiently be used for the manufacture of a medicament with vasodilating effect for the treatment of dysfunctions of the circulatory system, in particu- lar at the cardiovascular and coronary level. Accordingly, the compounds of the general formula (I), as well as their pharmaceutically accept- able salts, may be used, via the use of conventional pharmaceutical techniques, in the manufacture of me- dicaments which may be administrated by different routes. For example they may be administrated orally in form of pharmaceutically preparations such as tab- lets, capsules, syrups and suspensions. Parenterally in form of solutions or emulsions, etc. They may also be administrated topically in form of creams, pomades, balsams, etc., and transdermically for example through the use of patches or bandages. They may also be ap- plied directly in the rectum as suppositories. The preparations may comprise physiologically acceptable carriers, excipients, activators, chelating agents, stabilisators, etc. In case of injections there may be incorporated physiologically acceptable buffers, solu- bilizing agents or isotonics. The daily dose may be varied depending on the specific symptoms, the age, the body weight of the patients, the specific mode of ad- ministration, etc., and a daily normal dose for an adult person could be between 1 to 500 mg, and could be administrated as one dose only or divided into several doses during the day. In the working examples herein (vide infra) are described in details suitable processes to obtain various of the compounds according to the general for- mula (I) . In view of these examples, it is within the skilled persons general knowledge to obtain the com- pounds not explicitly exemplified herein via suitable modifications the of working examples herein. Consequently, the working examples herein should not be interpreted as limiting the scope of the invention, but solely as an additional detailed expli- cation, which guides the skilled person to a more deeply understanding of the invention. Examples The compounds obtained in the examples below are identified via its data in Infrared spectroscopy (IR), and/or Nuclear Magnetic Resonance spectroscopy of proton (1H-NMR) and of carbon 13 (l3C-MNR) . The IR spectra have been realized in film evaporated with CHC13 or in KBr tablet, in a PERKIN- ELMER FTIR model 1700 apparatus. The position of the most significant peaks are indicated in cm" . The Nuclear Magnetic Resonance spectra have been realized in a Varian Gemini-200 apparatus. In the spectra of 1H-NMR are indicated the working frequency and the solvent used to make the spectrum. The position of the signals is indicated in 8 (ppm), using as reference the signal of the protons of the solvent. The reference values are 7.24 ppm for the chloroform and 2.4 9 ppm for the deuterated dimethylsul- foxide. Within brackets are indicated the number of protons corresponding to each signal measured by elec- tronically integration and the type of signal is indi- cated using following abbreviations: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), sb (signal broad), sc (signal complex), d.e. D20 (disap- pears during realization of the spectrum after addition of some drops of deuterium water.) In the spectra of 13C-MNR are indicated the working frequency and the solvent on each spectrum. The position of the signals is indicated in 5 (ppm) , using as reference the signal of the protons of the solvent. The reference values are 77.00 ppm for the chloroform and 39.50 ppm for the deuterium dimethylsulfoxide. Further, there have been realized magnetic nuclear resonance experiments using the Attached Proton Test (APT). In the experimental part of the examples is used following abbreviations: AcOEt ethyl acetate DMSO-d6 dimethylsulfoxide hexa-deuterium EtOEt diethyl ether Example 1 Obtaining isosorbide 2-(2'- ethylthio)nico-tinate 5-mononitrate chlorhydrate. Step 1.- In a 50 mL glass flask, provided with a reflux refrigerator, closed with a CaCl2 tube, and magnetic agitation, 4.25 g (23.2 mmol) 2-ethylthionicotinate acid are dissolved in 20 mL of thionyl chloride (1.64 g/ml; 275.6 mmol). The reaction mixture is refluxed for 3.5 h. After this period, the mixture is cooled down and excess thionyl chloride is eliminated under reduced pressure while adding portions of toluene. After drying at reduced pressure, 4.67 g of a solid yellowish prod- uct corresponding to the acid chloride of interest are obtained. Yield: 100%. Step 2.- In a 50 ml glass flask, provided with magnetic agitation and reflux refrigerator, 4.67 g (23.2 mmol) of the acid chloride obtained in the step above are dissolved, under Ar atmosphere, in 25 ml pyridine. The solution is cooled down in a ice bath and 4.44 g (23.2 mmol) of isosorbide 5-mononitrate are added. The reac- tion mixture is agitated at room temperature under Ar atmosphere for 19 h. After this period the solvent is eliminated at reduced pressure. The residue is dis- solved in 50 mL of CHC13 and washed: first with 50 mL of water, secondly with 50 mL aqueous solution of 5% HC1 and once more with 50 mL water. The organic phase is dried over anhydrous MgSO4, filtered, and the solvent is eliminated at reduced pressure. After drying at reduced pressure, 7.25 g of the product of interest are ob- tained. Yield: 88%. Step 3.- In a 250 ml three neck glass flask, provided with a reflux refrigerator closed with a CaCl2 tube, magnetic agitation, and an addition funnel with a com- pensated pressure, 6.0 g (16.85 mmol) of the product obtained in the previous step are dissolved in 150 mL of EtOEt. The solution is agitated at room temperature and added, drop by drop, 30 mL of EtOEt solution satu- rated with HC1 (solution prior prepared bubbling HC1 gas directly into the EtOEt until saturation), produc- ing a white solid precipitate. The solid is filtered and washed with an excess of EtOEt and dried at reduced pressure. 6.55 g of the product of interest are ob- tained. Yield: 99%. 1H-NMR (200 MHz, DMSO-d6) : 10.26 (1H, s, d.e. D20, HC1), 8.60 (1H, dd, J=5 Hz, J=1. 8 Hz, CHar) , 8.20 (1H, dd, J=7.7 Hz, J=2 Hz, CHar) , 7.22 (1H, dd, J=3 Hz, J=8 Hz, CHar) , 5.43 (1H, sc, CH-ON02) , 5.30 (1 H,d, J=3 Hz, CH-O-CO) , 5.05 (1H, t, J=5.5 Hz, CH), 4.65 (1H, d, J=5 Hz, CH) , 4.20-3.80 (4H, sc, CH2), 3.17 (2H, q, J=7.6 Hz, CH2 -S) , 1.23 (3H, t, J=7.6 Hz, CH3) . 13C-NMR (50 MHz, DMSO-d6) : 164.06 (C=O) , 161.34 (Car- COO) , 152.88 (CHar), 139.63 (CHar) , 122.48 (Car- S), 119.13 (CHar), 86.19 (CH-ONO2) , 82.64 (CH) , 81.78 (CH), 78.10 (CH-O-CO), 72.90 (CH2) , 69.33 (CH2), 23.84 (CH2-S), 14.31 (CH3) . Step 1.- The same method as in step 2 of example 1 is used, applying as starting product the isosorbide 2- mononitrate. The product of interest is obtained at a chemical yield of 88%. Step 2.- In a 500 ml three neck glass flask provided with a reflux refrigerator closed with a CaCl2 tube, magnetic agitation, and a addition funnel with a com- pensated pressure, 7.0 g (19.66 mmol) of the product obtained in the former step are dissolved in a mixture of 200 mL of EtOEt + 100 mL de CH2C12. The solution is agitated at room temperature and added, drop by drop, 30 mL of EtOEt solution saturated with HC1 (solution prior prepared bubbling HCl gas directly into the EtOEt until saturatio), producing a white solid precipitate. The solid is filtered and washed with an excess of EtOEt and dried at reduced pressure. 7.05 g of the product of interest are obtained. Yield: 91%. 1H-NMR (200 MHz, DMSO-d6) : 8.63 (1H, dd, J=5 Hz, J=1.8 Hz, CHar), 8.33 (1H, sb, d.e. D2O, HC1), 8.23 (1H, dd, J=8 Hz, J=1.8 Hz, CHar) , 7.24 (1H, dd, J=3 Hz, J=7,8 Hz, CHar), 5.44 (1H, d, J=3.2 Hz, CH- O-CO), 5.33 (1H, sc, CHONO), 4.91 (1H, t, J=5.6 Hz, CH), 4.67 (1H, d, J=5.4 Hz, CH), 4.20-3.80 (4H, sc, CH2), 3.08 (2H, q, J=7.2 Hz, CH2-S) , 1.20 (3H, t, J=7.2 Hz CH3) l3C-NMR (50 MHz, DMSO-d6) : 163.74 (C=O) , 161.53 (Car- COO), 152.77 (CHar), 139.24 (CHar) , 122.05 (Car-S) , 119.01 (CHar), 86.65 (CH-ONO2) , 84.13 (CH) , 80.79 (CH), 74.48 (CH-O-CO), 70.78 (CH2-0) , 70.70 (CH2-O) , 23.67 (CH2), 14.14 (CH3) • Example 3.- Obtaining isosorbide 2-(2'~ mercapto)nicotinate 5-mononitrate {3) Step 1.- In a 100 mL glass flask, provided with a re- flux refrigerator closed with a CaCl2 tube and magnetic agitation, 3.0 g (19.35 mmol) of 2-mercaptonicotinic acid are suspended in 30 mL of thionyl chloride (1.64 g/ml; 431.4 mmol) . The mixture is left to reflux for 2h, observing the dissolution of the solid during this period. The mixture is cooled down and excess thionyl chloride is eliminated under reduced pressure while adding portions of toluene. After drying at reduced pressure, 3.35 g of a solid yellow-orange corresponding to the acid chloride of interest are obtained. Yield, 100%. Step 2.- In a 250 mL glass flask, provided with a re- flux refrigerator and magnetic agitation, 3.0 g (17.29 mmol) of the acid chloride obtained in the former step are suspended, under Ar atmosphere, in 75 mL of pyri- dine. The suspension is cooled down in an ice bath and 3.30 g (17.29 mmol) of isosorbide 5-mononitrate are added. The reaction mixture is left agitating at room temperature under Ar atmosphere for 19 h, a period of time wherein the mixture is getting dark. Once the re- action finished, the solvent is eliminated at reduced pressure. The residue is dissolved in 250 mL of CHCI= and washed: first with 250 mL of water, secondly with 250 mL aqueous solution of 5% HC1 and once more with 250 mL water. The organic phase is dried over anhydrous MgSO4, filtered, and the solvent is eliminated at re- duced pressure. After drying at reduced pressure, 5.45 g of a yellow solid are obtained, which are re- crystallized in isopropanol to obtain 4.83 g of a white solid which is reacted in acid medium for 20 rain, with triphenylphosphine (1:1.25 mol/mol) in methanol, with a 10% of water. The solvent is eliminated at reduced pre- sureand the residue is disolved in AcOEt, washing the solution with some water. The organic phase is dried and the solvent is eliminated at reduced presure, re- covering the product of interest by preparative chroma- tography. Yield: 35.7%. 1H-RMN (200 MHz, Cd3COCd3) : 7.90 (1H, dd, J=6.1 Hz, J=1.6 Hz, CHar), 7.70 (1H, dd, J=7 .2 Hz, J=l . 6 Hz, CHar) , 6.97 (1H, dd, J=6.4 Hz, J=7.2 Hz, CHar) , 5.63-5.55 (1H, sc, CH-ONO2), 5.38 (1H, d, J==3.4 Hz, CH-O-CO) , 5.09 (1H, t, J=5.1 Hz, CH), 4.75 (1H, d, J=4 . 8 Hz, CH), 4.20-3.85 (4H, sc, CH2) . IR (p.KBr):3438,2925,1735,1639,1571,1281,109s. Example 4.- Obtaining isosorbide 5-(2'- mercapto)nicotinate 2-mononitrate(4). In a 250 mL glass flask, provided with a reflux refrig- erator and magnetic agitation, 3.0 g (17.29 mmol) of the acid chloride obtained in step 1 of example 3 are suspended, under Ar atmosphere, in a mixture of 50 ml pyridine and 25 mL of CHCI3. The suspension is cooled down in an ice bath and 3.30 g (17.29 mmol) of isosor- bide 2-mononitrate are added. The reaction mixture is left agitating at room temperature under Ar atmosphere for 19 h, a period of time wherein the mixture is get- ting dark. Once the reaction finished, the solvent is eliminated at reduced pressure. The residue is dis- solved in 300 mL of CHC13 and washed: first with 300 mL of water, secondly with 300 mL aqueous solution of 5% HC1 and once more with 300 mL water. The organic phase is dried over anhydrous MgSO4, filtered, and the solvent is eliminated at reduced pressure. After drying at re- duced temperature, 5.10 g of a white-yellowish solid are obtained, which are re-crystallized in isopropanol to obtain 4.55 g of a white solid which is reacted in acid medium for 20 min. with triphenylphosphine (1:1.25 mol/mol) in methanol, with a 10% of water. The solvent is eliminated at reduced presure and the residue is disolved in AcOEt, washing the solution with some wa- ter. The organic phase is dried and the solvent is eliminated at reduced presure, recovering the product of interest by preparative chromatography. Yield: 37.6%. 1-RMN (200 MHz, Cd3COCd3) : 7.98 (1H, dd, J=4.2 Hz, J=1.0 Hz, CHar), 7.76 (1H, dd, J=4 .9 Hz, J=1.0 Hz, CHar) , 7.34 (1H, dd, J=4.5 Hz, J=4.8 Hz, CHar) , 5.50-5.36 (2H, sc, CH-ONO2+CH-O-CO) , 5.02 (1H, t, J=3.7 Hz, CH) , 4.74 (1H, d, J=3.4 Hz, CH), 4.20-3.90 (4H, sc, CH2) . IR (p.KBr):339s,2876,1727,1653,1631,1593,1291,1276. Example 5.- Obtaining 2-acetylmercaptoisosorbide 5- mononi-trate (5) . Step 1.- In a 1 L glass flask provided with a reflux refrigerator, an addition funnel with a compensated pressure, and magnetic agitation, 60 g (411 mmol) of isomanide, 88 g (461 mmol) of paratoluenesulfonyl chld- rine, 296 mL of CCI4, 33 mL of CH2C12 and 247 mL of H20 are mixed. An Ar atmosphere is made and a solution of 29.9 g (453 mmol) of 85% KOH is added, drop by drop, while maintaining the reaction temperature at 5° C. The period of time of the addition is 1 h 20 min. The re- sulting mixture is agitated at 5° C for 7 h. The solid is filtered and with 2 x 125 mL portions of H2O and dried at reduced pressure. The obtained solid is re-crystallized in 1200 mL of CCI4, hot filtered and the filtrate is left to cool down. The obtained crystals are filtered and washed yielding 54.5 g of a fraction A of the product of interest, monotosilate of isomanide. The, after the hot filtering, resulting solid is re-crystallized in 1000 mL of CCI4 obtaining 29.5 g of a fraction B of the product of interest. Step 2.- In a 500 mL glass flask provided with a reflux refrigerator and magnetic agitation, 22.7 g (76 mmol) of monotosilate of isomanide and 13.0 g (113 mmol) of potassium thioacetate are mixed in 113 mL of n-butanol. An Ar atmosphere is made and the reaction mixture is let to reflux for 1 h. The mixture is cooled down, fil- tered and washed with 200 mL ethanol and the solvents are eliminated at reduced pressure. 20 g of a solid are obtained. A thin layer chromatographic analysis with independent sample shows that the product of interest is not a major part of the crude. The obtained crude is treated with 300 mL of n-butanol and 40 mL of thioacetic acid and refluxed for 1 h. The mixture is left to cool down and filtered over a SiO2 layer. The solvents of the filtrate are evapo- rated at reduced pressure and a crude is obtained which is submitted to a Flash chromatography. For the chromatographic separation a mixture CHCl3/AcOEt 4:1 is used as eluent. A fraction of 4.14 g of 2-acetylmercaptoisosorbide is obtained, sufficiently pure to be used in the subsequent step of synthesis. Various fractions of product of interest are obtained with quite a lot of impurity. These last fractions are submitted to reverse phase preparative chromatography for purification of the wanted product. Step 3.- A nitrating mixture is prepared by addiing, slowly and carefully, 2.4 ml of 60% HNO3 into a mixture of 10 mL of acetic anhydride and 10 mL of acetic acid. The mixture is prepared at 0° C. In a 100 mL glass flask provided with a re- flux refrigerator and magnetic agitation, 2.51 g (12.3 mmol) of the product obtained in the former step are dissolved at 0° C in 14.5 mL of acetic acid and, after agitation for a while the nitrated mixture previously made is added drop by drop, for 20 minutes, while main- taining the temperature at 0° C. The reaction mixture is agitated for 2 h at 0° C, the crude is poured on 200 mL water, and the resulting mixture is extracted with 3 x 200 mL portions of AcOEt. Each of the three portions are washed separately with 2 x 220 mL portions of a saturated NaHCO3 solution and 200 mL of water. The ob- tained solution is dried on Na2SO4, filtered, and the solvents are eliminated at reduced pressure. 2.4 g of a crude are obtained which are submitted to a Flash Chro- matography using a mixture of CHCl3/AcOEt 25:1 as elu- ent. 2.0.8 g of product de interest are obtained. Yield: 68 %. 1H-NMR (200 MHz, CDC13) : 5.36-5.24 (1H, sc, CH- ONO2), 4.90-4.80 (1H, sc, CH), 4.44-4.37 (1H, sc, CH), 4.22-4.10 (1H, sc, CH) , 4.10-3.98 (2H, sc, CH2), 3.92-3.78 (2H, sc, CH2) , 2.33 (3H, s, CH3) . 13C-NMR (50 MHz, CDC13) : 194.48 (C=O) , 86.50 (CH- ONO2), 81.44 (CH), 81.22 (CH), 78.48 (CH2) , 69.25 (CH2), 45.92 (CH-S), 30.48 (CH3) . IR(cm-1): 300-2800, 1700, 1650, 1630, 1280, 1080, 960. Example 6 Obtaining isosorbide 2-(2'- methylthio)nico-tinate 5-mononitrate chlorhydrate (6) . In a 50 ml glass flask, provided with mag- netic agitation and reflux refrigerator, 2.00 g (10.7 mmol) of 2-methylthionicotinic acid chloride are sus- pended, under Ar atmosphere, in 12 ml pyridine. The mixture is cooled down in a ice bath and 2.04 g (10.7 mmol) of isosorbide 5-mononitrate are added. The reac- tion mixture is agitated at room temperature under Ar atmosphere for 15 h. After this period the solvent is eliminated at reduced pressure. The residue is dis- solved in 50 mL of CHC13 and washed: first with 50 mL of water, secondly with 50 mL aqueous solution of 5% HCl and once more with 50 mL water. The organic phase is dried over anhydrous MgSO4, filtered, and the solvent is eliminated at reduced pressure. After drying at reduced pressure, 2.80 g of the product of interest are ob- tained. Yield: 77%. 1H-RMN (200 MHz, DMSO-d6) : 8.68 (lH,dd,J=5 Hz, J=1.8 Hz, CHar), 8.22 (lH,dd,J=7.7 Hz, J=2 Hz, CHar) , 7.26 (lH,dd,J=3 Hz, J=8 Hz, CHar) , 5.54 (1H, td, J=2 Hz, J=6 Hz, CH-ON02), 5.34 (lH,d, J=3 Hz, CH-O-CO) , 5.06 (1H, t, J=5.5Hz, CH), 4.58 (1H, d, J=5 Hz, CH) , 4.18- 3.82 (4H, sc, CH2), 2.45 (3H, s, CH3-S) . 13C-RMN (50 MHz, DMSO-d6) : 163.91 (C=O) , 161.64 (Car-COO) , 152.80 (CHar)f 139.27 (CHar) , 122.20 (Car-S) , 118.83 (CHar), 85.97 (CH-ONO2) , 82.41 (CH) , 81.53 (CH) , 77.87 (CH-O-CO), 72.67 (CH2) , 69.07 (CH2) , 13.34 (CH3) . Example 7 Obtaining isosorbide 5-(2'- methylthio)nicotinate 2-mononitrate clorhydrate (2)• In a 50 ml glass flask, provided with mag- netic agitation and reflux refrigerator, 2.00 g (10.7 mmol) of 2-methylthionicotinic acid chloride are sus- pended, under Ar atmosphere, in 12 ml pyridine. The mixture is cooled down in a ice bath and 2.04 g (10.7 mmol) of isosorbide 5-mononitrate are added. The reac- tion mixture is agitated at room temperature under Ar atmosphere for 15 h. After this period the solvent is eliminated at reduced pressure. The residue is dis- solved in 50 mL of CHC13 and washed: first with 50 mL of water, secondly with 50 mL aqueous solution of 5% HC1 and once more with 50 mL water. The organic phase is dried over anhydrous MgSO4, filtered, and the solvent is eliminated at reduced pressure. After drying at reduced pressure, 2.75 g of the product of interest are ob- tained. Yield: 75%. 1H-RMN (200 MHz, DMSO-d6) : 8.90 (lH,dd,J=5 Hz, J=1.8 Hz, CHar), 8.27 (lH,dd,J=7.7 Hz, J=2 Hz, CHar) , 7.27 (lH,dd,J=3 Hz, J=7.8 Hz, CHar) , 5.42-5.31 (1H, sc, J=2 Hz, J=6 Hz, CH-ONO2), 5.60 (lH,d, J=3.2 Hz, CH-O- CO), 5.06 (1H, t, J=5.5Hz, CH) , 4.92 (1H, d, J=5. 6 Hz, CH), 4.10-3.88 (4H, sc, CH2) , 1.24 (3H, s, CH3-S) . 13C-RMN (50 MHz, DMSO-d6) : 163.71 (C=O) , 161.89 (Car-COO), 152.77 (CHar), 139.04 (CHar) , 121.92 (C.r-S) , 118.87 (CHar)/ 86.56 (CH-ONO2) , 84.05 (CH) , 80.69 (CH) 74.41 (CH-O-CO), 70.69 (CHZ) , 70.61 (CH2) , 13.37 (CH3) • Example 8.- Tests for vasodilatation. The method used in the assays is substan- tially the same as described in following references: * Furchgot, R.F. "Methods in nitric oxide re- search". Feelisch & Stamler eds. John Wiley &Sons, Chichester, England, pp 567-581. * Trongvanichnam, K, et al. Jpn J. Pharmacol. 1996; 71:167-173. * Salas, E., et al. Eur. J. Pharmacol. 1994; 258:47-55. The different compounds are tested at 5 dif- ferent concentrations, at a concentration range from 0.001 y 10 mM, using from 6 to 9 arterial rings for each compound. The obtained • results are compared to those from the isosorbide 5-mononitrate, which is used as reference product. The results are shown in table 1 below and are provided as CEso (concentration effective 50)/ which is the concentration of each of the tested compounds wherein there is produced a vasodilatation of 50% of the arterial ring previously contracted with 1 mM of Norepinephrine. As can be observed, the two compounds tested have a potent vasodilating activity, at least similar to that of the reference, and the compound 1 has a vasodilating activity superior to.that of thecreference product. Example 9.- Assay of tolerance. The different compound:, tested are subcutane- ously administrated to rats at a dose of 10 mg/Kg for three days, each eight hours, and the assay is then done ex vivo to test the capacity to vasodilate the ar- terial segments of the rats after the subcutaneous ad- ministration of the compound. The method followed is substantially the same as described in following references:. ? De Garavilla, L., et al. Eur. J. Pharmacol. 1996; 313:89-96. • Keith, R.A., et al. J. Pharmacol. Exp. Ther. 1982; 221:525-531. The different compounds are tested at 5 dif- ferent concentrations, at a concentration, range from 0.001 to 10 mM, using from 6 to 9 arterial rings for each compound. The obtained results are compared to those from the isosorbide 5-mononitrate, which is used as reference product, and with those obtained from the animals wherein there have not been administrated any compound. The results obtained, also shown as CE50, are shown in table 2 It should be understood that a compound de- velops tolerance when the CE50 of the product in the vascular rings of the animals which have been submitted to administration of the compound, as specified above, is superior to the CE50 of the compound in the vascular rings of the animals which have not been submitted to administration of the compound. The CE50 of isosorbide 5-mononitrcite in the group of animals wherein said compound was adminis- trated was seven times superior as compared to that of the not treated animals. which indicate a strong developments of tolerance for the reference product. On the contrary, for the two compounds tested, 1 y 5, which form part of the object of the invention, the CE50 obtained for both of them are significantly less, which indicate a development of tolerance very inferior as compared to the reference product. Further, for the compound 5 the development of tolerance is practically null under these test condi- tions. CLAIMS 5 i. A derivative of isosorbide moponitrate, or a pharmaceutically acceptable salt thereof, having the following general formula (I) 10 15 wherein one of A and B represents -ONO2 and the other represents 20 wherein: either (i) Z is a sulphur atom and R is an optionally substituted C1-C4 alkyl group, an optionally substituted aryl group or an 25 optionally substituted aralkyl group; or (ii) Z is an oxygen atom and R is the group 30 wherein R1 is hydrogen or an optionally substituted C1-C4 alkyl group, an optionally substituted aryl group or an optionally 35 substituted aralkyl group. 2. A compound as claimed in claim 1, wherein Z is a sulphur atom and R is a C1 - C4 alkyl group. 3. A compound as claimed in claim 1, wherein Z is an oxygen atom, and R1 is a hydrogen atom or a C1 - C4 alkyl group. 4. A compound as claimed in any preceding claim, wherein B is the -ONO2 group. 5. The compound as claimed in claim 1, which is isosorbide 2 - (2 ' -ethylthio) nicotinate 5-mononitrate or a pharmaceutically acceptable salt thereof. 6. The compound as claimed in claim 1, which is isosorbide 5 - (2 ' -ethylthio) nicotinate 2-mononitrate or a pharmaceutically acceptable salt thereof. 7. The compound as claimed in claim 1, which is isosorbide 2 - (2 ' -mercapto) nicotinate 5-mononitrate or a pharmaceutically acceptable salt thereof. 8. The compound as claimed in claim 1, which is isosorbide 5 - (2 ' -mercapto) nicotinate 2-mononitrate or a pharmaceutically acceptable salt thereof. 9. The compound as claimed in claim 1, which is 2 - Acetylmercaptoisosorbide 5-mononitrate. 10. A pharmaceutical composition comprising a pharmaceutically effective dose of a compound, as claimed in any of the claims 1 to 9, or pharmaceutically acceptable salts thereof, as active ingredient, optionally together with physiologically acceptable carriers, excipients, activators, chelating agents and/or stabilizers, said composition being meant for the treatment of cardiovascular or coronary dysfunctions, and having vasodilating effect for the treatment of dysfunctions of circulatory system. Novel derivatives of isosorbide mononitrate and its pharmaceutically acceptable salts, which have vasodi- 5 lating activity with a reduced effect of tolerance, of the general formula (I) in which A and B individually represent any of the groups 10 -ONO2 and -Z-CO-R, wherein Z is an oxygen atom or sul- phur atom and R is an alkyl C1 - C4 group, an aryl group or an aralkyl group, eventually substituted, or the group 15 in which R1 is hydrogen, or an alkyl C1 - C4 group, an aryl group or an aralkyl group, eventually substituted, with the proviso that one of A or B is always -ONO2, but never both of them at the same time, when Z is an sul- phur atom R is an alkyl C1 - C4 group, an aryl group or an 20 aralkyl group, eventually substituted, and when Z is an oxygen atom R is the group

Full Text

DERIVATIVES OF ISOSORBIDE MONONITRATE AND PHARMACEUTICAL
COMPOSITION CONTAINING THE SAME FOR TREATMENT OF
CARDIOVASCULAR OR CORONARY DYSFUNCTIONS, OR FOR
TREATMENT OF DYSFUNCTIONS OF CIRCULATORY SYSTEM.
Field of the invention
The present invention relates to novel derivatives of isosorbide mononitrate
which have a potent vasodilating activity and which, at the same time, have a
significantly reduced tolerance, and in particular, pharmaceutical composition
containing the same for treatment of cardiovascular or coronary dysfunctions, or for
treatment of dysfunctions of circulatory system.
Background art
The nitric acid esters of organic: compounds,
common known as nitrated organic compounds, are known
and have been used as vasodilating agents. Within
these, the usefulness of mono and di-nitrated isosor-
bide is well known, and further there have been de-
scribed compounds with vascular and coronary activities
based on substitution reactions of the free hydroxyl
group of isosorbide mononitrate- For example, the US-A-
4891373 patent describes derivatives of aminepropanol
corresponding to the formulas
for the treatment of the angina pectoris and systemic
and pulmonary hypertension.
The US-A-5665766 patent describes the isosor-
bide 5-mononitrate 2-acetylsalicylate, of formula
as well as its platelets anti-aggregating activity.
One of the principal problems of the nitrated
organic compounds mentioned above resides on the fact
that these are quite sensible in relation to the phe-
nomena known as tachyphylaxy or tolerance, which re-
lates to that its effect on the organism decreases dur-
ing prolonged treatment, and it is then required to
sensitively elevate the administrated doses in a gradu-
ated manner or otherwise to perform a pharmacologically
wash out.
It is also known that one way of reducing the
tolerance of the nitrated organic compounds consists of
introducing a thiol group in the molecule, for example
by use of sulphur containing amino acids. The European
patent EP-B-0362575 describes nitrated organic com-
pounds with incorporated Cysteine and/ mainly, Methion-
ine molecules.
The patent application WO-A-92/04337 de-
scribes organic nitrated derivatives of the ring of the
thiazolidine with vasodilating activity and a reduced
tolerance.
The patent application WO-A-93/03037 de-
scribes an enormously amount of different nitrated or-
ganic vasodilating compounds, with reduced tolerance,
of highly variable structures. Within these are in-
cluded generically, i.e. without specifying nor de-
scribing one single specific product, derivatives of
isosorbide mononitrate according to following structure
in which R5 represents a hydrogen atom, a C1-C6 alkyl
group, a phenyl, etc.
The nitrated organic compounds described in
the documents mentioned above do not in itself solve
the problems originating from the tolerance of the ni-
trated organic compounds, since these still have prob-
lems in relation to low vasodilating activity, high
tolerance, etc.. Accordingly, it is still necessary to
develop novel nitrated organic compounds which have a
high vasodilating activity combined with a more de-
creased level of tolerance being maintained persis-
tently.
Summary of the invention
An object of the invention is a novel type of
compounds, derivatives of isosorbide mononitrate, which
are capable of providing a potent vasodilating effect
and which at the same time show a small or null toler-
ance effect.
A further object of the present invention re-
lates to the use of the novel derivatives of isosorbide
mononitrate for the manufacture of a medicament for the
treatment of disorders related to dysfunctions of the
circulatory system, in particular at the level of the
coronary system.
Detailed description of the invention
According to the present invention there is provided a
derivative of isosorbide mononitrate, or a
pharmaceutically acceptable salt thereof, having the
following general formula (I)
wherein one of A and B represents -ONO2 and the other
represents
wherein:
either (i) Z is a sulphur atom and R is an
optionally substituted C1-C4 alkyl group, an
optionally substituted aryl group or an
optionally substituted aralkyl group;
or (ii) Z is an oxygen atom and R is the group
v/herein R1 is hydrogen or an optionally
substituted C1-C4 alkyl group, an optionally
substituted aryl group or an optionally
substituted aralkyl group.
Within the novel derivatives of the invention
it is preferred that when Z is a sulphur atom, R is. a
short chain C1-C4 alkyl group, and when Z is an oxygen
atom, R1 is a hydrogen atom or a short chain C1-C4 alkyl
group. More preferably, within above mentioned crite-
ria, B is the -ONO2 group, i.e. the compounds wherein
the nitrate ester is at position 5 in the ring-shaped
system of the isosorbide.
The preferences mentioned above should not in
any way be considered as limiting the scope of the ob-
ject of the present invention.
In case R1 is hydrogen the compounds of the
invention could be represented as any of its two
tautomers
and both of the tautomer structures should be consid-
ered as within the object of the invention.
Examples of specific compounds within the ob-
ject of the invention.could be following:
isosorbide 2- (2'-ethylthio)nicotinate 5-mononitrate, of
formula
isosorbide 5-(2'-ethylthio)nicotinate 2-mononitrate, of
formula
isosorbide 2-(2'-mercapto)nicotinate 5-mononitrate, of
formula
isosorbide 5-{2'-mercapto)nicotinate 2-mononitrate, of
formula
2-acetylmercaptoisosorbide 5-mononitrate, of formula
isosorbide 2-(2'-methylthio)nicotinate 5-mononitrate,
of formula
isosorbide 5-(2'-ethylthio)nicotinate 2-mononitrate, of
formula
as well as the pharmaceutically acceptable salt of
these, in particular their chlorhydrates.
The compounds 1 and its chlorhydrate and the
compound 5 are particularly preferred.
The compounds of the present invention can be
obtained by techniques of esterification using known or
accessible starting products described in the basic or-
ganic chemical literature known to the skilled person,
for example the publications of Chemical Abstracts Ser-
vice, the Beilstein Encyclopedia of organic products,
or in any other appropriate publication available at
university libraries.
For example, when Z is an oxygen atom the
compounds may be obtained from isosorbide or the corre-
sponding isosorbide mononitrate through a reaction of
esterification of these with the corresponding carbox-
ylic acid or an activated derivative of this, for exam-
ple an acid chloride, an anhydride, an active ester,
etc. If the starting product is isosorbide, it will be
necessary finishing with a further step consisting of
nitrating the free hydroxyl group of the isosorbide, a
thing which is not necessary if there is started from
any of the isosorbide mononitrates in position 5 or in
position 2 of the ring-shaped structure of said com-
pound .
When R1 is hydrogen, these compounds have
a thiol group which can be unintentionally oxidized
producing disulphur dimers. In this case, the dimers
can be reverted to the corresponding monomers by reac-
tion with triphenylphosphine in water, as described in
R. Humphrey (1964), Analytical Chem,36,1812 y
L.E.Overman (1974), Synthesis, 59.
When Z is a sulphur atom the situation is
very similar since it is enough to start from the cor-
responding thiocarboxylic acid, in stead of the carbox-
ylic acid mentioned above, and to use techniques well
known to the expert for the formation of the thioester
bond. On the other hand, if any of the reactions imply
the epimerization of a chiral center, there may be used
as a starting compound the adequate enantiomer of the
isosorbide, for example the isomanide.
The tests performed demonstrate that the
novel isosorbide mononitrate derivatives of the inven-
tion show a vasodilating activity comparable, as a
minimum, with that of the isosorbide mononitrate by it-
self, and in some cases highly superior. Further, they
manifest a significant inferior tolerance as compared
to that observed with said compound and in some cases
it approaches practically null.
Consequently, the compounds of the invention
may very efficiently be used for the manufacture of a
medicament with vasodilating effect for the treatment
of dysfunctions of the circulatory system, in particu-
lar at the cardiovascular and coronary level.
Accordingly, the compounds of the general
formula (I), as well as their pharmaceutically accept-
able salts, may be used, via the use of conventional
pharmaceutical techniques, in the manufacture of me-
dicaments which may be administrated by different
routes.
For example they may be administrated orally
in form of pharmaceutically preparations such as tab-
lets, capsules, syrups and suspensions. Parenterally in
form of solutions or emulsions, etc. They may also be
administrated topically in form of creams, pomades,
balsams, etc., and transdermically for example through
the use of patches or bandages. They may also be ap-
plied directly in the rectum as suppositories. The
preparations may comprise physiologically acceptable
carriers, excipients, activators, chelating agents,
stabilisators, etc. In case of injections there may be
incorporated physiologically acceptable buffers, solu-
bilizing agents or isotonics. The daily dose may be
varied depending on the specific symptoms, the age, the
body weight of the patients, the specific mode of ad-
ministration, etc., and a daily normal dose for an
adult person could be between 1 to 500 mg, and could be
administrated as one dose only or divided into several
doses during the day.
In the working examples herein (vide infra)
are described in details suitable processes to obtain
various of the compounds according to the general for-
mula (I) . In view of these examples, it is within the
skilled persons general knowledge to obtain the com-
pounds not explicitly exemplified herein via suitable
modifications the of working examples herein.
Consequently, the working examples herein
should not be interpreted as limiting the scope of the
invention, but solely as an additional detailed expli-
cation, which guides the skilled person to a more
deeply understanding of the invention.
Examples
The compounds obtained in the examples below
are identified via its data in Infrared spectroscopy
(IR), and/or Nuclear Magnetic Resonance spectroscopy of
proton (1H-NMR) and of carbon 13 (l3C-MNR) .
The IR spectra have been realized in film
evaporated with CHC13 or in KBr tablet, in a PERKIN-
ELMER FTIR model 1700 apparatus. The position of the
most significant peaks are indicated in cm" .
The Nuclear Magnetic Resonance spectra have
been realized in a Varian Gemini-200 apparatus.
In the spectra of 1H-NMR are indicated the
working frequency and the solvent used to make the
spectrum. The position of the signals is indicated in 8
(ppm), using as reference the signal of the protons of
the solvent. The reference values are 7.24 ppm for the
chloroform and 2.4 9 ppm for the deuterated dimethylsul-
foxide. Within brackets are indicated the number of
protons corresponding to each signal measured by elec-
tronically integration and the type of signal is indi-
cated using following abbreviations: s (singlet), d
(doublet), t (triplet), dd (doublet of doublets), sb
(signal broad), sc (signal complex), d.e. D20 (disap-
pears during realization of the spectrum after addition
of some drops of deuterium water.)
In the spectra of 13C-MNR are indicated the
working frequency and the solvent on each spectrum. The
position of the signals is indicated in 5 (ppm) , using
as reference the signal of the protons of the solvent.
The reference values are 77.00 ppm for the chloroform
and 39.50 ppm for the deuterium dimethylsulfoxide.
Further, there have been realized magnetic
nuclear resonance experiments using the Attached Proton
Test (APT).
In the experimental part of the examples is
used following abbreviations:
AcOEt ethyl acetate
DMSO-d6 dimethylsulfoxide hexa-deuterium
EtOEt diethyl ether
Example 1 Obtaining isosorbide 2-(2'-
ethylthio)nico-tinate 5-mononitrate
chlorhydrate.
Step 1.- In a 50 mL glass flask, provided with a reflux
refrigerator, closed with a CaCl2 tube, and magnetic
agitation, 4.25 g (23.2 mmol) 2-ethylthionicotinate
acid are dissolved in 20 mL of thionyl chloride (1.64
g/ml; 275.6 mmol). The reaction mixture is refluxed for
3.5 h. After this period, the mixture is cooled down
and excess thionyl chloride is eliminated under reduced
pressure while adding portions of toluene. After drying
at reduced pressure, 4.67 g of a solid yellowish prod-
uct corresponding to the acid chloride of interest are
obtained. Yield: 100%.
Step 2.- In a 50 ml glass flask, provided with magnetic
agitation and reflux refrigerator, 4.67 g (23.2 mmol)
of the acid chloride obtained in the step above are
dissolved, under Ar atmosphere, in 25 ml pyridine. The
solution is cooled down in a ice bath and 4.44 g (23.2
mmol) of isosorbide 5-mononitrate are added. The reac-
tion mixture is agitated at room temperature under Ar
atmosphere for 19 h. After this period the solvent is
eliminated at reduced pressure. The residue is dis-
solved in 50 mL of CHC13 and washed: first with 50 mL of
water, secondly with 50 mL aqueous solution of 5% HC1
and once more with 50 mL water. The organic phase is
dried over anhydrous MgSO4, filtered, and the solvent is
eliminated at reduced pressure. After drying at reduced
pressure, 7.25 g of the product of interest are ob-
tained. Yield: 88%.
Step 3.- In a 250 ml three neck glass flask, provided
with a reflux refrigerator closed with a CaCl2 tube,
magnetic agitation, and an addition funnel with a com-
pensated pressure, 6.0 g (16.85 mmol) of the product
obtained in the previous step are dissolved in 150 mL
of EtOEt. The solution is agitated at room temperature
and added, drop by drop, 30 mL of EtOEt solution satu-
rated with HC1 (solution prior prepared bubbling HC1
gas directly into the EtOEt until saturation), produc-
ing a white solid precipitate. The solid is filtered
and washed with an excess of EtOEt and dried at reduced
pressure. 6.55 g of the product of interest are ob-
tained. Yield: 99%.
1H-NMR (200 MHz, DMSO-d6) : 10.26 (1H, s, d.e. D20,
HC1), 8.60 (1H, dd, J=5 Hz, J=1. 8 Hz, CHar) , 8.20
(1H, dd, J=7.7 Hz, J=2 Hz, CHar) , 7.22 (1H, dd,
J=3 Hz, J=8 Hz, CHar) , 5.43 (1H, sc, CH-ON02) , 5.30
(1 H,d, J=3 Hz, CH-O-CO) , 5.05 (1H, t, J=5.5 Hz,
CH), 4.65 (1H, d, J=5 Hz, CH) , 4.20-3.80 (4H, sc,
CH2), 3.17 (2H, q, J=7.6 Hz, CH2 -S) , 1.23 (3H, t,
J=7.6 Hz, CH3) .
13C-NMR (50 MHz, DMSO-d6) : 164.06 (C=O) , 161.34 (Car-
COO) , 152.88 (CHar), 139.63 (CHar) , 122.48 (Car-
S), 119.13 (CHar), 86.19 (CH-ONO2) , 82.64 (CH) ,
81.78 (CH), 78.10 (CH-O-CO), 72.90 (CH2) , 69.33
(CH2), 23.84 (CH2-S), 14.31 (CH3) .
Step 1.- The same method as in step 2 of example 1 is
used, applying as starting product the isosorbide 2-
mononitrate. The product of interest is obtained at a
chemical yield of 88%.
Step 2.- In a 500 ml three neck glass flask provided
with a reflux refrigerator closed with a CaCl2 tube,
magnetic agitation, and a addition funnel with a com-
pensated pressure, 7.0 g (19.66 mmol) of the product
obtained in the former step are dissolved in a mixture
of 200 mL of EtOEt + 100 mL de CH2C12. The solution is
agitated at room temperature and added, drop by drop,
30 mL of EtOEt solution saturated with HC1 (solution
prior prepared bubbling HCl gas directly into the EtOEt
until saturatio), producing a white solid precipitate.
The solid is filtered and washed with an excess of
EtOEt and dried at reduced pressure. 7.05 g of the
product of interest are obtained. Yield: 91%.
1H-NMR (200 MHz, DMSO-d6) : 8.63 (1H, dd, J=5 Hz,
J=1.8 Hz, CHar), 8.33 (1H, sb, d.e. D2O, HC1), 8.23
(1H, dd, J=8 Hz, J=1.8 Hz, CHar) , 7.24 (1H, dd,
J=3 Hz, J=7,8 Hz, CHar), 5.44 (1H, d, J=3.2 Hz, CH-
O-CO), 5.33 (1H, sc, CHONO), 4.91 (1H, t, J=5.6 Hz,
CH), 4.67 (1H, d, J=5.4 Hz, CH), 4.20-3.80 (4H, sc,
CH2), 3.08 (2H, q, J=7.2 Hz, CH2-S) , 1.20 (3H, t,
J=7.2 Hz CH3)
l3C-NMR (50 MHz, DMSO-d6) : 163.74 (C=O) , 161.53 (Car-
COO), 152.77 (CHar), 139.24 (CHar) , 122.05 (Car-S) ,
119.01 (CHar), 86.65 (CH-ONO2) , 84.13 (CH) , 80.79
(CH), 74.48 (CH-O-CO), 70.78 (CH2-0) , 70.70 (CH2-O) ,
23.67 (CH2), 14.14 (CH3) •
Example 3.- Obtaining isosorbide 2-(2'~
mercapto)nicotinate 5-mononitrate {3)
Step 1.- In a 100 mL glass flask, provided with a re-
flux refrigerator closed with a CaCl2 tube and magnetic
agitation, 3.0 g (19.35 mmol) of 2-mercaptonicotinic
acid are suspended in 30 mL of thionyl chloride (1.64
g/ml; 431.4 mmol) . The mixture is left to reflux for
2h, observing the dissolution of the solid during this
period. The mixture is cooled down and excess thionyl
chloride is eliminated under reduced pressure while
adding portions of toluene. After drying at reduced
pressure, 3.35 g of a solid yellow-orange corresponding
to the acid chloride of interest are obtained. Yield,
100%.
Step 2.- In a 250 mL glass flask, provided with a re-
flux refrigerator and magnetic agitation, 3.0 g (17.29
mmol) of the acid chloride obtained in the former step
are suspended, under Ar atmosphere, in 75 mL of pyri-
dine. The suspension is cooled down in an ice bath and
3.30 g (17.29 mmol) of isosorbide 5-mononitrate are
added. The reaction mixture is left agitating at room
temperature under Ar atmosphere for 19 h, a period of
time wherein the mixture is getting dark. Once the re-
action finished, the solvent is eliminated at reduced
pressure. The residue is dissolved in 250 mL of CHCI=
and washed: first with 250 mL of water, secondly with
250 mL aqueous solution of 5% HC1 and once more with
250 mL water. The organic phase is dried over anhydrous
MgSO4, filtered, and the solvent is eliminated at re-
duced pressure. After drying at reduced pressure, 5.45
g of a yellow solid are obtained, which are re-
crystallized in isopropanol to obtain 4.83 g of a white
solid which is reacted in acid medium for 20 rain, with
triphenylphosphine (1:1.25 mol/mol) in methanol, with a
10% of water. The solvent is eliminated at reduced pre-
sureand the residue is disolved in AcOEt, washing the
solution with some water. The organic phase is dried
and the solvent is eliminated at reduced presure, re-
covering the product of interest by preparative chroma-
tography. Yield: 35.7%.
1H-RMN (200 MHz, Cd3COCd3) : 7.90 (1H, dd, J=6.1 Hz,
J=1.6 Hz, CHar), 7.70 (1H, dd, J=7 .2 Hz, J=l . 6 Hz, CHar) ,
6.97 (1H, dd, J=6.4 Hz, J=7.2 Hz, CHar) , 5.63-5.55 (1H,
sc, CH-ONO2), 5.38 (1H, d, J==3.4 Hz, CH-O-CO) , 5.09 (1H,
t, J=5.1 Hz, CH), 4.75 (1H, d, J=4 . 8 Hz, CH), 4.20-3.85
(4H, sc, CH2) .
IR (p.KBr):3438,2925,1735,1639,1571,1281,109s.
Example 4.- Obtaining isosorbide 5-(2'-
mercapto)nicotinate 2-mononitrate(4).
In a 250 mL glass flask, provided with a reflux refrig-
erator and magnetic agitation, 3.0 g (17.29 mmol) of
the acid chloride obtained in step 1 of example 3 are
suspended, under Ar atmosphere, in a mixture of 50 ml
pyridine and 25 mL of CHCI3. The suspension is cooled
down in an ice bath and 3.30 g (17.29 mmol) of isosor-
bide 2-mononitrate are added. The reaction mixture is
left agitating at room temperature under Ar atmosphere
for 19 h, a period of time wherein the mixture is get-
ting dark. Once the reaction finished, the solvent is
eliminated at reduced pressure. The residue is dis-
solved in 300 mL of CHC13 and washed: first with 300 mL
of water, secondly with 300 mL aqueous solution of 5%
HC1 and once more with 300 mL water. The organic phase
is dried over anhydrous MgSO4, filtered, and the solvent
is eliminated at reduced pressure. After drying at re-
duced temperature, 5.10 g of a white-yellowish solid
are obtained, which are re-crystallized in isopropanol
to obtain 4.55 g of a white solid which is reacted in
acid medium for 20 min. with triphenylphosphine (1:1.25
mol/mol) in methanol, with a 10% of water. The solvent
is eliminated at reduced presure and the residue is
disolved in AcOEt, washing the solution with some wa-
ter. The organic phase is dried and the solvent is
eliminated at reduced presure, recovering the product
of interest by preparative chromatography. Yield:
37.6%.
1-RMN (200 MHz, Cd3COCd3) : 7.98 (1H, dd, J=4.2 Hz,
J=1.0 Hz, CHar), 7.76 (1H, dd, J=4 .9 Hz, J=1.0 Hz, CHar) ,
7.34 (1H, dd, J=4.5 Hz, J=4.8 Hz, CHar) , 5.50-5.36 (2H,
sc, CH-ONO2+CH-O-CO) , 5.02 (1H, t, J=3.7 Hz, CH) , 4.74
(1H, d, J=3.4 Hz, CH), 4.20-3.90 (4H, sc, CH2) .
IR
(p.KBr):339s,2876,1727,1653,1631,1593,1291,1276.
Example 5.- Obtaining 2-acetylmercaptoisosorbide 5-
mononi-trate (5) .
Step 1.- In a 1 L glass flask provided with a reflux
refrigerator, an addition funnel with a compensated
pressure, and magnetic agitation, 60 g (411 mmol) of
isomanide, 88 g (461 mmol) of paratoluenesulfonyl chld-
rine, 296 mL of CCI4, 33 mL of CH2C12 and 247 mL of H20
are mixed. An Ar atmosphere is made and a solution of
29.9 g (453 mmol) of 85% KOH is added, drop by drop,
while maintaining the reaction temperature at 5° C. The
period of time of the addition is 1 h 20 min. The re-
sulting mixture is agitated at 5° C for 7 h. The solid
is filtered and with 2 x 125 mL portions of H2O and
dried at reduced pressure.
The obtained solid is re-crystallized in 1200
mL of CCI4, hot filtered and the filtrate is left to
cool down. The obtained crystals are filtered and
washed yielding 54.5 g of a fraction A of the product
of interest, monotosilate of isomanide.
The, after the hot filtering, resulting solid
is re-crystallized in 1000 mL of CCI4 obtaining 29.5 g
of a fraction B of the product of interest.
Step 2.- In a 500 mL glass flask provided with a reflux
refrigerator and magnetic agitation, 22.7 g (76 mmol)
of monotosilate of isomanide and 13.0 g (113 mmol) of
potassium thioacetate are mixed in 113 mL of n-butanol.
An Ar atmosphere is made and the reaction mixture is
let to reflux for 1 h. The mixture is cooled down, fil-
tered and washed with 200 mL ethanol and the solvents
are eliminated at reduced pressure. 20 g of a solid are
obtained.
A thin layer chromatographic analysis with
independent sample shows that the product of interest
is not a major part of the crude.
The obtained crude is treated with 300 mL of
n-butanol and 40 mL of thioacetic acid and refluxed for
1 h. The mixture is left to cool down and filtered over
a SiO2 layer. The solvents of the filtrate are evapo-
rated at reduced pressure and a crude is obtained which
is submitted to a Flash chromatography.
For the chromatographic separation a mixture
CHCl3/AcOEt 4:1 is used as eluent. A fraction of 4.14 g
of 2-acetylmercaptoisosorbide is obtained, sufficiently
pure to be used in the subsequent step of synthesis.
Various fractions of product of interest are obtained
with quite a lot of impurity. These last fractions are
submitted to reverse phase preparative chromatography
for purification of the wanted product.
Step 3.- A nitrating mixture is prepared by addiing,
slowly and carefully, 2.4 ml of 60% HNO3 into a mixture
of 10 mL of acetic anhydride and 10 mL of acetic acid.
The mixture is prepared at 0° C.
In a 100 mL glass flask provided with a re-
flux refrigerator and magnetic agitation, 2.51 g (12.3
mmol) of the product obtained in the former step are
dissolved at 0° C in 14.5 mL of acetic acid and, after
agitation for a while the nitrated mixture previously
made is added drop by drop, for 20 minutes, while main-
taining the temperature at 0° C. The reaction mixture
is agitated for 2 h at 0° C, the crude is poured on 200
mL water, and the resulting mixture is extracted with 3
x 200 mL portions of AcOEt. Each of the three portions
are washed separately with 2 x 220 mL portions of a
saturated NaHCO3 solution and 200 mL of water. The ob-
tained solution is dried on Na2SO4, filtered, and the
solvents are eliminated at reduced pressure. 2.4 g of a
crude are obtained which are submitted to a Flash Chro-
matography using a mixture of CHCl3/AcOEt 25:1 as elu-
ent. 2.0.8 g of product de interest are obtained. Yield:
68 %.
1H-NMR (200 MHz, CDC13) : 5.36-5.24 (1H, sc, CH-
ONO2), 4.90-4.80 (1H, sc, CH), 4.44-4.37 (1H, sc,
CH), 4.22-4.10 (1H, sc, CH) , 4.10-3.98 (2H, sc,
CH2), 3.92-3.78 (2H, sc, CH2) , 2.33 (3H, s, CH3) .
13C-NMR (50 MHz, CDC13) : 194.48 (C=O) , 86.50 (CH-
ONO2), 81.44 (CH), 81.22 (CH), 78.48 (CH2) , 69.25
(CH2), 45.92 (CH-S), 30.48 (CH3) .
IR(cm-1): 300-2800, 1700, 1650, 1630, 1280, 1080,
960.
Example 6 Obtaining isosorbide 2-(2'-
methylthio)nico-tinate 5-mononitrate
chlorhydrate (6) .
In a 50 ml glass flask, provided with mag-
netic agitation and reflux refrigerator, 2.00 g (10.7
mmol) of 2-methylthionicotinic acid chloride are sus-
pended, under Ar atmosphere, in 12 ml pyridine. The
mixture is cooled down in a ice bath and 2.04 g (10.7
mmol) of isosorbide 5-mononitrate are added. The reac-
tion mixture is agitated at room temperature under Ar
atmosphere for 15 h. After this period the solvent is
eliminated at reduced pressure. The residue is dis-
solved in 50 mL of CHC13 and washed: first with 50 mL of
water, secondly with 50 mL aqueous solution of 5% HCl
and once more with 50 mL water. The organic phase is
dried over anhydrous MgSO4, filtered, and the solvent is
eliminated at reduced pressure. After drying at reduced
pressure, 2.80 g of the product of interest are ob-
tained. Yield: 77%.
1H-RMN (200 MHz, DMSO-d6) : 8.68 (lH,dd,J=5 Hz,
J=1.8 Hz, CHar), 8.22 (lH,dd,J=7.7 Hz, J=2 Hz, CHar) ,
7.26 (lH,dd,J=3 Hz, J=8 Hz, CHar) , 5.54 (1H, td, J=2 Hz,
J=6 Hz, CH-ON02), 5.34 (lH,d, J=3 Hz, CH-O-CO) , 5.06
(1H, t, J=5.5Hz, CH), 4.58 (1H, d, J=5 Hz, CH) , 4.18-
3.82 (4H, sc, CH2), 2.45 (3H, s, CH3-S) .
13C-RMN (50 MHz, DMSO-d6) : 163.91 (C=O) , 161.64
(Car-COO) , 152.80 (CHar)f 139.27 (CHar) , 122.20 (Car-S) ,
118.83 (CHar), 85.97 (CH-ONO2) , 82.41 (CH) , 81.53 (CH) ,
77.87 (CH-O-CO), 72.67 (CH2) , 69.07 (CH2) , 13.34 (CH3) .
Example 7 Obtaining isosorbide 5-(2'-
methylthio)nicotinate 2-mononitrate
clorhydrate (2)•
In a 50 ml glass flask, provided with mag-
netic agitation and reflux refrigerator, 2.00 g (10.7
mmol) of 2-methylthionicotinic acid chloride are sus-
pended, under Ar atmosphere, in 12 ml pyridine. The
mixture is cooled down in a ice bath and 2.04 g (10.7
mmol) of isosorbide 5-mononitrate are added. The reac-
tion mixture is agitated at room temperature under Ar
atmosphere for 15 h. After this period the solvent is
eliminated at reduced pressure. The residue is dis-
solved in 50 mL of CHC13 and washed: first with 50 mL of
water, secondly with 50 mL aqueous solution of 5% HC1
and once more with 50 mL water. The organic phase is
dried over anhydrous MgSO4, filtered, and the solvent is
eliminated at reduced pressure. After drying at reduced
pressure, 2.75 g of the product of interest are ob-
tained. Yield: 75%.
1H-RMN (200 MHz, DMSO-d6) : 8.90 (lH,dd,J=5 Hz,
J=1.8 Hz, CHar), 8.27 (lH,dd,J=7.7 Hz, J=2 Hz, CHar) ,
7.27 (lH,dd,J=3 Hz, J=7.8 Hz, CHar) , 5.42-5.31 (1H, sc,
J=2 Hz, J=6 Hz, CH-ONO2), 5.60 (lH,d, J=3.2 Hz, CH-O-
CO), 5.06 (1H, t, J=5.5Hz, CH) , 4.92 (1H, d, J=5. 6 Hz,
CH), 4.10-3.88 (4H, sc, CH2) , 1.24 (3H, s, CH3-S) .
13C-RMN (50 MHz, DMSO-d6) : 163.71 (C=O) , 161.89
(Car-COO), 152.77 (CHar), 139.04 (CHar) , 121.92 (C.r-S) ,
118.87 (CHar)/ 86.56 (CH-ONO2) , 84.05 (CH) , 80.69 (CH)
74.41 (CH-O-CO), 70.69 (CHZ) , 70.61 (CH2) , 13.37 (CH3) •
Example 8.- Tests for vasodilatation.
The method used in the assays is substan-
tially the same as described in following references:
* Furchgot, R.F. "Methods in nitric oxide re-
search". Feelisch & Stamler eds. John Wiley
&Sons, Chichester, England, pp 567-581.
* Trongvanichnam, K, et al. Jpn J. Pharmacol. 1996;
71:167-173.
* Salas, E., et al. Eur. J. Pharmacol. 1994;
258:47-55.
The different compounds are tested at 5 dif-
ferent concentrations, at a concentration range from
0.001 y 10 mM, using from 6 to 9 arterial rings for
each compound. The obtained • results are compared to
those from the isosorbide 5-mononitrate, which is used
as reference product.
The results are shown in table 1 below and
are provided as CEso (concentration effective 50)/ which
is the concentration of each of the tested compounds
wherein there is produced a vasodilatation of 50% of
the arterial ring previously contracted with 1 mM of
Norepinephrine.
As can be observed, the two compounds tested
have a potent vasodilating activity, at least similar
to that of the reference, and the compound 1 has a
vasodilating activity superior to.that of thecreference
product.
Example 9.- Assay of tolerance.
The different compound:, tested are subcutane-
ously administrated to rats at a dose of 10 mg/Kg for
three days, each eight hours, and the assay is then
done ex vivo to test the capacity to vasodilate the ar-
terial segments of the rats after the subcutaneous ad-
ministration of the compound.
The method followed is substantially the same
as described in following references:.
? De Garavilla, L., et al. Eur. J. Pharmacol. 1996;
313:89-96.
• Keith, R.A., et al. J. Pharmacol. Exp. Ther.
1982; 221:525-531.
The different compounds are tested at 5 dif-
ferent concentrations, at a concentration, range from
0.001 to 10 mM, using from 6 to 9 arterial rings for
each compound. The obtained results are compared to
those from the isosorbide 5-mononitrate, which is used
as reference product, and with those obtained from the
animals wherein there have not been administrated any
compound.
The results obtained, also shown as CE50, are
shown in table 2
It should be understood that a compound de-
velops tolerance when the CE50 of the product in the
vascular rings of the animals which have been submitted
to administration of the compound, as specified above,
is superior to the CE50 of the compound in the vascular
rings of the animals which have not been submitted to
administration of the compound.
The CE50 of isosorbide 5-mononitrcite in the
group of animals wherein said compound was adminis-
trated was seven times superior as compared to that of
the not treated animals.

which indicate a strong developments of tolerance for
the reference product. On the contrary, for the two
compounds tested, 1 y 5, which form part of the object
of the invention, the CE50 obtained for both of them are
significantly less, which indicate a development of
tolerance very inferior as compared to the reference
product. Further, for the compound 5 the development of
tolerance is practically null under these test condi-
tions.
CLAIMS
5
i. A derivative of isosorbide moponitrate, or a
pharmaceutically acceptable salt thereof, having the
following general formula (I)
10
15 wherein one of A and B represents -ONO2 and the other
represents
20 wherein:
either (i) Z is a sulphur atom and R is an
optionally substituted C1-C4 alkyl group, an
optionally substituted aryl group or an
25 optionally substituted aralkyl group;
or (ii) Z is an oxygen atom and R is the group
30
wherein R1 is hydrogen or an optionally
substituted C1-C4 alkyl group, an optionally
substituted aryl group or an optionally
35 substituted aralkyl group.
2. A compound as claimed in claim 1, wherein Z is a sulphur atom and R is a C1 - C4
alkyl group.
3. A compound as claimed in claim 1, wherein Z is an oxygen atom, and R1 is a
hydrogen atom or a C1 - C4 alkyl group.
4. A compound as claimed in any preceding claim, wherein B is the -ONO2
group.
5. The compound as claimed in claim 1, which is isosorbide 2 - (2 ' -ethylthio)
nicotinate 5-mononitrate or a pharmaceutically acceptable salt thereof.
6. The compound as claimed in claim 1, which is isosorbide 5 - (2 ' -ethylthio)
nicotinate 2-mononitrate or a pharmaceutically acceptable salt thereof.
7. The compound as claimed in claim 1, which is isosorbide 2 - (2 ' -mercapto)
nicotinate 5-mononitrate or a pharmaceutically acceptable salt thereof.
8. The compound as claimed in claim 1, which is isosorbide 5 - (2 ' -mercapto)
nicotinate 2-mononitrate or a pharmaceutically acceptable salt thereof.
9. The compound as claimed in claim 1, which is 2 - Acetylmercaptoisosorbide
5-mononitrate.
10. A pharmaceutical composition comprising a pharmaceutically effective dose
of a compound, as claimed in any of the claims 1 to 9, or pharmaceutically acceptable
salts thereof, as active ingredient, optionally together with physiologically acceptable
carriers, excipients, activators, chelating agents and/or stabilizers, said composition
being meant for the treatment of cardiovascular or coronary dysfunctions, and having
vasodilating effect for the treatment of dysfunctions of circulatory system.
Novel derivatives of isosorbide mononitrate and its
pharmaceutically acceptable salts, which have vasodi-
5 lating activity with a reduced effect of tolerance, of
the general formula (I)
in which A and B individually represent any of the
groups
10 -ONO2 and -Z-CO-R, wherein Z is an oxygen atom or sul-
phur atom and R is an alkyl C1 - C4 group, an aryl group
or an aralkyl group, eventually substituted, or the
group
15 in which R1 is hydrogen, or an alkyl C1 - C4 group, an
aryl group or an aralkyl group, eventually substituted,
with the proviso that one of A or B is always -ONO2, but
never both of them at the same time, when Z is an sul-
phur atom R is an alkyl C1 - C4 group, an aryl group or an
20 aralkyl group, eventually substituted, and when Z is an
oxygen atom R is the group

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Patent Number

223406

Indian Patent Application Number

IN/PCT/2001/00395/KOL

PG Journal Number

37/08

Publication Date

12-Sep-2008

Grant Date

10-Sep-2008

Date of Filing

09-Apr-2001

Name of Patentee

LACER S.A.

Applicant Address

CALLE CERDENA, 350, E-08025 BARCELONA

Inventors:

#	Inventor's Name	Inventor's Address
1	PUBILL COY FRANCISCO	GRAN VIA CORTS CATALANES 1039,E-08020 BARCELONA
2	CABEZA LLORENTE LYDIA	CALLE VILAPICINA, 8,E-08031 BARCELONA
3	CARBO BANUS MARCEL LI	CALLE ALBERT PINYOL,2-4 E-08029 BARCELONA
4	CERDA RIUDAVETS JUAN ANTONIO	CALLE LACY, 94,E-08202 SABADELL
5	NEGRIE ROFES CRISTINA	DE SAVORNIN LOHMANPLEIN 2, NL-2314 EV LEIDEN
6	FERRER SISO ALICIA	CALLE CONCEPCION ARENAL 49-51, E-08027, BARCELONA
7	RADOMSKI MAREK, W	UNIVERSITY OF ALBERTA DEPARTMENT OF PHARMA-COLOGY, 9-50 MEDICAL SCIENCE BUILDING, 114 STREET-89 AVENUE EDMONTON, ALBERTA T6G 2M7
8	SALAS PEREZ-RASILLA EDUARDO	CALLE CARTAGENA 258-260 E-08025 BARCELONA
9	MARTINEZ BONNIN JUAN	PASSEIG DEL BORN, 27-29 E-08003, BARCELONA
10	REPOLLES MOLINER JOSE	CALLE PARIS, 46-48, E-08029 BARCELONA

PCT International Classification Number

C07D 493/04

PCT International Application Number

PCT/ES99/00316

PCT International Filing date

1999-10-04

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	P 9802076	1998-10-07	Spain