NICOTINAMIDE MONONUCLEOTIDE DERIVATIVES FOR THE TREATMENT OF ARRHYTHMIA

Abstract

A method for treating arrythmia in subject in need thereof, which includes administering to the subject a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt and/or solvate thereof;

##STR00001##

in which X, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, Y, and are as described in the claims.

Claims

1.-10. (canceled)

11. A method for treating arrythmia in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a compound of formula (I)× ##STR00068## or a pharmaceutically acceptable salt and/or solvate thereof, wherein: X is selected from O, CH.sub.2, S, Se, CHF, CF.sub.2 and C═CH.sub.2; R.sub.1 is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are selected, independently of one another, from H, halogen, azido, cyano, hydroxyl, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 thio-alkyl, C.sub.1-C.sub.12 heteroalkyl, C.sub.1-C.sub.12 haloalkyl and OR; wherein R is selected from H, C.sub.1-C.sub.12 alkyl, C(O)(C.sub.1-C.sub.12)-alkyl, C(O)NH(C.sub.1-C.sub.12)-alkyl, C(O)O(C.sub.1-C.sub.12)-alkyl, C(O)-aryl, C(O)(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)NH(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)O(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl and C(O)CHR.sub.AANH.sub.2; wherein Ra is a side chain selected from a proteinogenic amino acid; R.sub.6 is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.7 is selected from P(O)R.sub.9R.sub.10, P(S)R.sub.9R.sub.10 and ##STR00069## wherein R.sub.9 and R.sub.10 are selected, independently of one another, from OH, OR.sub.11, NHR.sub.13, NR.sub.13R.sub.14, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.12 aryl, (C.sub.5-C.sub.12)-aryl-(C.sub.1-C.sub.8)-alkyl, (C.sub.1-C.sub.8)-alkyl-(C.sub.5-C.sub.12)-aryl, (C.sub.1-C.sub.8)-heteroalkyl, (C.sub.3-C.sub.8)-heterocycloalkyl, (C.sub.5-C.sub.12)-heteroaryl and NHCR.sub.αR.sub.α′C(O)R.sub.12; wherein: R.sub.11 is selected from C.sub.1-C.sub.10 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.12 aryl, (C.sub.1-C.sub.10)-alkyl-(C.sub.5-C.sub.12)-aryl, C.sub.5-C.sub.12 substituted aryl, C.sub.1-C.sub.10 heteroalkyl, C.sub.1-C.sub.10 haloalkyl, —(CH.sub.2).sub.mC(O)(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mOC(O)(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mOC(O)O(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mSC(O)(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mC(O)O(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mC(O)O(C.sub.1-C.sub.15)-alkyl-aryl; wherein m is an integer selected from 1 to 8; and P(O)(OH)OP(O)(OH).sub.2; an internal or external counter-ion; R.sub.12 is selected from hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.1-C.sub.10 haloalkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.12 aryl, (C.sub.1-C.sub.4)-alkyl-(C.sub.5-C.sub.12)-aryl and C.sub.5-C.sub.12 heteroaryl; wherein said aryl or heteroaryl groups are optionally substituted by one or two groups selected from halogen, trifluoromethyl, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy and cyano; R.sub.13 and R.sub.14 are selected independently from H, C.sub.1-C.sub.8 alkyl and (C.sub.1-C.sub.8)-alkyl-(C.sub.5-C.sub.12)-aryl; R.sub.α and R.sub.α′ selected independently, from hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.1-C.sub.10 thio-alkyl, C.sub.1-C.sub.10 hydroxylalkyl, (C.sub.1-C.sub.10)-alkyl-(C.sub.5-C.sub.12)-aryl, C.sub.5-C.sub.12 aryl, —(CH.sub.2).sub.3NHC(═NH)NH.sub.2, (1H-indol-3-yl)-methyl, (1H-imidazol-4-yl)-methyl and a side chain selected from a proteinogenic or non-proteinogenic amino acid; wherein said aryl groups are optionally substituted by a group selected from hydroxyl, C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.6 alkoxy, halogen, nitro and cyano; or R.sub.9 and R.sub.10, with the phosphorus atoms to which they are bonded, form a 6-member-ring, wherein —R.sub.9-R.sub.10— represents —CH.sub.2—CH.sub.2—CHR— or —O—CH.sub.2—CH.sub.2—CHR—O—; wherein R is selected from hydrogen, C.sub.5-C.sub.6 aryl and C.sub.5-C.sub.6 heteroaryl; wherein said aryl or heteroaryl groups are optionally substituted by one or two groups selected from halogen, trifluoromethyl, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy and cyano; X′ is selected from O, CH.sub.2, S, Se, CHF, CF.sub.2 and C═CH.sub.2; R.sub.1′ is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.2′, R.sub.3′, R.sub.4′ and R.sub.5′ are selected, independently of one another, from H, halogen, azido, cyano, hydroxyl, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 thio-alkyl, C.sub.1-C.sub.12 heteroalkyl, C.sub.1-C.sub.12 haloalkyl and OR; wherein R is selected from H, C.sub.1-C.sub.12 alkyl, C(O)(C.sub.1-C.sub.12)-alkyl, C(O)NH(C.sub.1-C.sub.12)-alkyl, C(O)O(C.sub.1-C.sub.12)-alkyl, C(O)-aryl, C(O)(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)NH(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)O(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl and C(O)CHR.sub.AANH.sub.2; wherein RA is a side chain selected from a proteinogenic amino acid; R.sub.6′ is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.8′ is selected from H, OR, NHR.sub.15′, NR.sub.15′R.sub.16′, NH—NHR.sub.15′, SH, CN, N.sub.3 and halogen; wherein R.sub.15′ and R.sub.16′ are selected, independently of one another, from H, C.sub.1-C.sub.8 alkyl and C.sub.1-C.sub.8 alkyl-aryl; Y′ is selected from CH, CH.sub.2, C(CH.sub.3).sub.2 and CCH.sub.3; n is an integer selected from 1 to 3; represents a single or a double bond according to Y′; and represents the alpha or beta anomer according to the position of R.sub.1′; R.sub.8 is selected from H, OR, NHR.sub.15, NR.sub.15R.sub.16, NH—NHR.sub.15, SH, CN, N.sub.3 and halogen; wherein R is selected from H and C.sub.1-C.sub.12, alkyl, and R.sub.15 and R.sub.16 are selected, independently of one another, from H, C.sub.1-C.sub.8 alkyl and C.sub.1-C.sub.8 alkyl-aryl and —CHR.sub.AACO.sub.2H wherein R.sub.AA is a side chain selected from a proteinogenic or non-proteinogenic amino acid; Y is selected from CH, CH.sub.2, C(CH.sub.3).sub.2 and CCH.sub.3; represents a single or a double bond according to Y; and represents the alpha or beta anomer according to the position of R.sub.1.

12. The method according to claim 11, wherein X represents oxygen.

13. The method according to claim 11, wherein R.sub.1 and R.sub.6 each represent hydrogen.

14. The method according to claim 11, wherein R.sub.2, R.sub.3, R.sub.4 and R.sub.5 each represent, independently of one another, hydrogen or OH.

15. The method according to claim 11, wherein Y represents CH.

16. The method according to claim 11, wherein Y represents CH.sub.2.

17. The method according to claim 11, wherein R.sub.7 represents P(O)R.sub.9R.sub.10 or ##STR00070## wherein R.sub.9 and R.sub.10 are as defined in claim 1 and X′ is oxygen; R.sub.1′ and R.sub.6′ each represent hydrogen; R.sub.2′, R.sub.3′, R.sub.4′ and R.sub.5′ are independently selected from hydrogen and OH; R.sub.8′ is NH.sub.2; Y′ is selected from CH and CH.sub.2; n is equal to 2; represents a single or a double bond according to Y′; and represents the alpha or beta anomer according to the position de R.sub.1′.

18. The method according to claim 11, wherein R.sub.7 represents P(O)(OH).sub.2.

19. The method according to claim 11, wherein the compound of formula (I) is selected from the list of compounds consisting of: ##STR00071## ##STR00072## and pharmaceutically acceptable salts and solvates thereof.

20. The method according to claim 11, wherein the type of arrhythmia is selected from the group consisting of bradycardia, tachycardia, auricular fibrillation, ventricular tachycardia and ventricular fibrillation.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0267] FIG. 1 is a histogram showing the incidence (FIG. 1A) and duration (FIG. 1B) of the ventricular tachycardia analysed during the ischaemia.

[0268] FIG. 2 is a histogram showing the incidence (FIG. 2A) and duration (FIG. 2B) of the ventricular fibrillation analysed during the ischaemia.

[0269] FIG. 3 is a histogram showing the incidence (FIG. 3A) and duration (FIG. 3B) of the ventricular tachycardia analysed during the reperfusion.

[0270] FIG. 4 is a histogram showing the incidences (FIG. 4A) and duration (FIG. 4B) of the ventricular fibrillation analysed during the reperfusion, as well as the number of ventricular fibrillations (FIG. 4C) during this period.

[0271] FIG. 5 is a histogram showing the mortality rate of rats treated with a saline solution, the compound of formula I-C and cariporide.

[0272] FIG. 6 is a histogram showing the heart rate 5 days after the injection of the carrier or DOX (20 mg/kg). *** p<0.001: t-test or Mann-Whitney test-DOX mice treated with the carrier vs control mice, $$$ p<0.001: Unidirectional ANOVA followed by a post-hoc Dunnett test or Kruskal-Wallis test followed by a post-hoc Dunn test-DOX treated mice with the carrier vs DOX mice treated with NMN analogues (180 mg/kg) or a carrier.

EXAMPLES

[0273] The present invention will be better understood on reading the following examples which illustrate the invention in a non-limiting manner.

I. Synthesis of the Compounds of the Invention

1. Material and Methods

[0274] All the chemicals were obtained from commercial suppliers and users without further purification. Thin layer chromatography was carried out on plastic sheets of TLC silica gel 60 F254 (layer thickness 0.2 mm) from Merck. Purification by column chromatography was carried out on the silica gel 60 (70-230 mesh ASTM, Merck). The melting points were determined either on a digital apparatus (Electrothermal IA 8103) and are not corrected, or on a WME Kofler bench (Wagner & Munz). The IR, .sup.1H, .sup.19F and .sup.13C NMR spectra confirmed the structure of all the compounds. The IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer and the NMR spectra were recorded, using CDCl.sub.3, CD.sub.3CN, D.sub.2O or DMSO-d.sub.6 as solvent, on a BRUKER AC 300 or 400 spectrometer at 300 or 400 MHz for the .sup.1H spectrum, 75 or 100 MHz for the .sup.13C spectrum and 282 or 377 MHz for the .sup.19F spectrum. The chemical shifts (δ) were expressed in parts per million with respect to the signal, indirectly (i) to CHCl.sub.3 (δ 7.27) for .sup.1H and (ii) to CDCl.sub.3 (δ 77.2) for .sup.13C and directly (iii) to CFCl.sub.3 (internal standard) (δ 0) for .sup.19F. The chemical shifts are given in ppm and the multiplicities of peaks are designated as follows: s, singlet; br s, wide singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quadruplet; quint, quintuplet; m, multiplet. High resolution mass spectra (HRMS) were obtained from the “Service central d'analyse de Solaize” (Centre national de la recherche scientifique) and have been recorded on a Waters spectrometer, using electrospray ionisation-TOF (ESI-TOF).

General Procedure

Step 1: Synthesis of the Compound of Formula A-1

[0275] The compound of formula D (1.0 equiv.) is dissolved in dichloromethane. The nicotinamide of formula E (1.50 equiv.) and TMSOTf (1.55 equiv.) are added at ambient temperature. The mixture is heated with reflux and stirred until the reaction is achieved. The mixture is cooled to ambient temperature and filtered. The filtrate is concentrated to dryness to give tetraacetate A-1.

Step 2: Synthesis of the Compound of Formula I-A

[0276] The tetraacetate A-1 is dissolved in methanol and cooled to −10° C. 4.6 M ammonia in methanol (3.0 equivalents) at −10° C. is added and the mixture is stirred at this temperature until the reaction is complete. Dowex HCR (H+) resin is added to a pH of 6-7. The reaction mixture is heated to 0° C. and filtered. The resin is washed with a mixture of methanol and acetonitrile. The filtrate is concentrated to dryness. The residue is dissolved in acetonitrile and concentrated to dryness. The residue is dissolved in acetonitrile to give a solution of the compound of formula I-A.

Step 3: Synthesis of the Compound of Formula I-A′

[0277] The crude solution of the compound of formula I-A in acetonitrile is diluted with trimethyl phosphate (10.0 equivalents). The acetonitrile is distilled under vacuum and the mixture is cooled to −10° C. Phosphorus oxychloride (4.0 equivalents) is added at −10° C. and the mixture is stirred at −10° C. until the reaction is ended.

Step 4 and S: Synthesis of the Compound of Formula I-C

[0278] The mixture is hydrolysed by the addition of a 50/50 mixture of acetonitrile and water, followed by the addition of methyl tert-butyl ether. The mixture is filtered and the solid is dissolved in water. The aqueous solution is neutralised by the addition of sodium bicarbonate and extracted with dichloromethane. The aqueous layer is concentrated to dryness in order to give the crude compound of formula I-C, which is purified on a DOWEX 50w×8 column with elution in water followed by a silica gel chromatograph column.

II. Evaluation of the Compounds of the Invention in a Model of Arrhythmia Induced by Ischaemia-Reperfusion

[0279] The aim of this study was to evaluate whether the administration of NAD precursor can attenuate the appearance of arrhythmia in an ischaemia-reperfusion rat.

[0280] All the procedures have been carried out in accordance with the Guidelines for the care and use of laboratory animals (revised in 1996 and 2011, 2010/63/UE) and with French law.

1. Material and Methods

1.1. Materials

1.1.1. Products Tested

[0281]

TABLE-US-00003 TABLE 3 Name Compound of formula I-C Concentration in the 37 mg/ml (5 mL/kg, iv) formulation Carrier 0.9% NaCl Required quantity 1200 mg Appearance White powder Storage conditions Ambient temperature Preparation protocol Weigh the powder and dissolve it in the carrier. Storage conditions for the 4° C. formulation Frequency of preparation The day of the experiment Shelf life of the formulation 24 hours

1.1.2. Reference Product

[0282]

TABLE-US-00004 TABLE 4 Name Cariporide Concentration 12 mg/ml (5 mL/kg, iv) Storage conditions 4° C. Preparation protocol Solutions with 12 mg/ml in 100% DMSO were prepared and aliquoted. For each rat, one aliquot was used and dissolved in order to obtain a solution with 0.06 mg/ml in 0.5% DMSO. Storage conditions Solution with 12 mg/ml in 100% DMSO at −20° C. for the formulation Solution 0.06 mg/ml in 0.5% DMSO at ambient temperature. Frequency of Daily (for the dilution) preparation Shelf life of One day the formulation

1.1.3. Carrier

[0283] Saline Solution

1.1.4. Animals Used

[0284]

TABLE-US-00005 TABLE 5 Species Rat Race Sprague Dawley Sex Male Age / Number 36 animals have been included gradually as a function of the mortality rate (~30%) in order to obtain at least 8 evaluable animals per treatment group at the end of the experimental phase. Weight 250-300 g Supplier JANVIER SAS Justification Males were selected to avoid the hormonal cycle.

[0285] The animals were housed in ventilated breeding cages which were GR900-enriched (905 CM.sup.2, Tecniplast) throughout the entire acclimatisation period and the experimental phase. The animal cages were provided with sufficient nesting material to completely cover the animals (Sizzle-Nest: unbleached brown kraft paper from Bio-service), wooden sticks (aspen bricks from Bio-service). The animal cage bedding (particles of poplar wood, without chemical treatment, pre-comminuted, de-dusted, sieved and dehydrated popular wood, without chemical treatment, from SDS DIETEX) was changed at least once per week. They were housed in groups of 2 animals with a standard 12-hour light cycle (lights off at 20:00), at 22±2° C. and 55±10% relative humidity.

[0286] An acclimatisation period of at least 5 days was applied.

[0287] Throughout this phase, SDS water and tap water were supplied ad libitum.

[0288] Arrhythmias were obtained in an acute model of ischaemia-reperfusion in rats.

1.1.5. Treatment

[0289] Dosage Regimen and Test Groups:

TABLE-US-00006 TABLE 6 Group Nbr/ Administration Start of No. group treatment Route Dose volume/rate treatment 1 n = 12 Carrier IV 0.9% Bolus 30 minutes (Saline 5 mL/kg before solution) ischaemia 2 n = 11 Compound 185 mg/kg I-C 3 n = 9 Cariporide 0.3 mg/kg 5 minutes before ischaemia

1.2. Method

1.2.1. Venous Catheterisation and Blood Pressure/ECG Recording

[0290] The rats were first anaesthetised, then intubated and mechanically ventilated, before being prepared for surgery.

[0291] Catheters were then placed in the carotid artery in order to measure the arterial pressure and in the caudal vein for the perfusion of drugs. The arterial catheter consists in a catheter filled with fluid which sends the pressure to a transducer located nearby. The mean arterial pressure is calculated electronically and recorded continuously.

[0292] A three-lead electrocardiogram (ECG) was recorded throughout the procedure by means of needle electrodes attached to the limbs.

[0293] The modifications to the ECG, mean arterial pressure (MAP) and the heart rate were measured before and during the occlusion-reperfusion period. The definitions of the arrhythmias are based on those described in the Lambeth conventions (Walker and al., The Lambeth conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion, Cardiovascular Research, 1988, 22(7), 447-455). The ectopic activity has been categorized as a single ventricular premature beat (VPB), a ventricular tachycardia (VT, four or more consecutive VPBs) or a ventricular fibrillation (VF, inability to distinguish individual QRS complexes and to measure the rate). The arterial pressure traces were referred to in order to confirm which type of ectopic activity was produced, in particular to distinguish between polymorphic VT and VF. When the first is produced, the arterial pressure is generally still pulsatile whereas with VF, the arterial pressure falls rapidly towards zero and is no longer pulsatile. The VF can be sustained or can spontaneously return to a normal sinusoidal rhythm in rats. In all the experiments, the incidences of TV and VF as well as the duration of the ventricular tachycardia were noted.

1.2.2. Arrhythmia Induced by Ischaemia-Reperfusion in Anaesthetised Rats

[0294] The heart was then exposed by a left intercostal thoracotomy. After opening the pericardium, a 6.0 silk stitch was placed around the left coronary artery on a polypropylene tube in order to form a collar. The hearts were subjected to an ischaemia for 7 minutes by tightening the suture 5 minutes after the treatment. Finally, the collar occluder was released in order to allow the reperfusion of the myocardial tissue for 10 minutes. At the end of the reperfusion period, the rats were euthanised by cervical dislocation while still anaesthetised.

1.2.3. Data Analysis

[0295] The means± the standard error on the mean are presented.

[0296] The statistical analysis was carried out using the GraphPad Prism 5 software. The Fisher test was used to analyse the differences between the incidences of arrhythmias, while the chi-squared test was used to compare the incidence of mortality. The Kruskall-Wallis test was used to analyse the differences between the duration of the tachycardia and the ventricular fibrillations and the difference in the number of ventricular fibrillations.

[0297] For all the tests, p<0.05 will be considered as significant.

2. Results and Discussion

[0298] The rats were treated by the intravenous route with a saline solution, a compound of formula I-C at 185 mg/kg (30 minutes before the ischaemia) or cariporide at 0.3 mg/kg (5 minutes before the ischaemia). The animals were subjected to a 7-minute ischaemia and a 10-minute reperfusion. Throughout the experimental phase, the arterial pressure and ECG profiles were recorded continuously. Blood was sampled 5 minutes after the end of the reperfusion and the heart was weighed.

[0299] FIG. 1 shows the incidence (FIG. 1A) and duration (FIG. 1B) of the ventricular tachycardia during the ischaemia.

[0300] FIG. 2 shows the incidence (FIG. 2A) and duration (FIG. 2B) of the fibrillation, analysed during the ischaemia.

[0301] FIG. 3 shows the incidence (FIG. 3A) and duration (FIG. 3B) of the ventricular tachycardia during the reperfusion.

[0302] FIG. 4 shows the incidence (FIG. 4A) and duration (FIG. 4B) of fibrillation during the reperfusion, as well as the number of ventricular fibrillations (FIG. 4C) during this period.

[0303] In the group of the carrier, the ischaemia has caused a ventricular tachycardia in half of the animals (duration: 4.7±2.4 s) and a ventricular fibrillation was observed in one rat, whereas the reperfusion has caused a ventricular tachycardia in all the animals (duration: 8.9 t 3.1% of the duration of the reperfusion), and a ventricular fibrillation in 75% of the animals (duration: 17.8±8.4% of the reperfusion period with approximately two events per animal).

[0304] Moreover, as shown in FIG. 5, for 25% of the animals, the ventricular arrhythmias during reperfusion were irreversible and have led to death.

[0305] As envisaged, the preventative treatment with cariporide removed the ventricular tachycardia and fibrillation during the ischaemia. During reperfusion, cariporide has had no beneficial effect on the incidence of ventricular tachycardia, but the duration has had a tendency to be shorter with respect to the group of carriers. Cariporide has removed the ventricular fibrillation and mortality during the reperfusion period.

[0306] During the ischaemia, the preventative treatment by a compound of formula I-C has made it possible to significantly reduce the incidence and duration of ventricular tachycardia (incidence: approximately 9%, duration: 0.2±0.2 s) and to remove the ventricular fibrillation. As with cariporide, the compound of formula I-C has had no effect on the incidence of ventricular tachycardia during reperfusion, but has appeared to reduce the duration. Although it is not statistically significant, a tendency to reduce the incidence of ventricular fibrillation was observed during reperfusion in the group treated by the compound of formula I-C compared to the carrier (incidence: approximately 55% and duration: 4.0±2.3% of the duration of the reperfusion with approximately one event per animal). Finally and above all, the compound of formula I-C has removed mortality during the reperfusion period.

[0307] The weight of the heart was a similar between the groups.

3. Conclusion

[0308] Ischaemia-reperfusion has been used, previously for generating an infarction and studying the protective effect of Nicotinamide Mononucleotide (NMN) against this infarction (Journal of Cardiovascular Pharmacology and Therapeutics, 2019, pp. 1-11; J.Mol Cell Cardiol. 2018, Vol. 121, pp. 155-162).

[0309] In this model, the infarction is induced by an ischaemia of at least 30 minutes and a reperfusion of at least 60 minutes. These severe conditions make it possible to model the infarction, which is generally triggered by the obstruction of an artery which supplies the heart with blood and therefore with oxygen, causing death of the muscle cells of the heart over a more or less extended region. It has been shown that NMN protects the heart and enables the patient to recover better after the infarction.

[0310] Here, much less severe conditions, in other words a 7-minute ischaemia followed by a 10-minute reperfusion can induce a ventricular tachycardia and a (non-fatal and fatal) fibrillation. Cariporide has removed the arrhythmias during the ischaemic period, as well as the ventricular fibrillation and the mortality during the reperfusion. The compound of formula I-C has removed the tachycardia and the ventricular fibrillations during the ischaemic period, has had a tendency to prevent ventricular fibrillation during reperfusion and has removed mortality during the reperfusion.

III. Evaluation of the Compounds of the Invention in a Model of Arrhythmia Induced by Doxorubicin

[0311] The goal of this study was to evaluate the effects of an i.p. administration of compounds I-C, I-J and I-K, at 180 mg/kg, on arrhythmias induced by doxorubicin.

1. Material and Methods

1.1.Materials

1.1.1. Animals

[0312] 76 male mice, 8 weeks old on arrival, were obtained from Janvier Labs, Le Genest St Isle, 53941 St Berthevin, France. Each animal was identified with an electronic chip.

1.1.2. Products

[0313] The compounds I-C, I-J and I-K were tested and stored at +4° C. before use. The carrier was a physiological buffer.

1.2. Method

1.2.1. Preparation of Formulations

[0314] The powder of compounds I-C, I-J and I-K (180 mg/kg) was dissolved in the carrier (the solution was used at ambient temperature for a maximum of 1 day).

1.2.2. Arrhythmia Induced by Doxorubicin

[0315] Arrhythmias due to a cardiotoxicity were induced by a single intraperitoneal injection of doxorubicin (DOX) at 20 mg/kg. Doxorubicin was prepared at 2 mg/mL and the administration volume was 10 mL/kg. The control group received an injection of saline solution.

1.2.3. Experimental Groups

[0316] Description of the Groups:

[0317] Group 1: saline solution+Carrier

[0318] Group 2: Doxorubicin (20 mg/kg)+Carrier

[0319] Group 3: Doxorubicin (20 mg/kg)+Compound I-C (180 mg/kg)

[0320] Group 4: Doxorubicin (20 mg/kg)+Compound I-J (180 mg/kg)

[0321] Group 5: Doxorubicin (20 mg/kg)+Compound I-K (180 mg/kg)

[0322] Distribution of Groups:

[0323] Each group comprises 14 to 24 mice. As indicated in the regulations relating to non-clinical laboratory studies, the groups of test and control animals have been kept under identical conditions. The envisaged duration of the study was 11 days.

1.2.4. Induction with Doxorubicin

[0324] On Day 0, the mice received an administration of DOX (20 mg/kg) by the intraperitoneal route.

1.2.5. Treatment

[0325] The treatment with compounds I-C, I-J and I-K was initiated after 5 days before the injection of DOX, once per day from Day 5 to Day 0.

[0326] The mice were treated i.p with compounds I-C, I-J and I-K 30 minutes before the injection of DOX.

[0327] The mice were treated i.p with compounds I-C, I-J and I-K during the duration of the experiment (J0 to J5) once per day. The last injection took place 24 hours before the sacrifice.

1.2.6. Evaluation of Cardiac Function by Electrocardiogram

[0328] An electrocardiogram (ECG) was produced 5 days after the injection of doxorubicin in the anaesthetised animals (isoflurane 1.5-2%) by non-invasive, two-dimensional echocardiography (VF16-5 probe, Siemens, Acuson NX3 Elite).

[0329] In particular, the heart rate was evaluated during the ECG:

2. Results and Discussion

[0330] FIG. 6 shows the heart rate 5 days after injection of saline solution or DOX (20 mg/kg).

[0331] The doxorubicin considerably reduced the heart rate compared with the control mice (365.1±23.9 bpm vs 525.6±19.8 bpm respectively). The treatments with compounds I-C, I-J and I-K caused an increase in heart rates, the compound I-J significantly improving this parameter (470.1±18.8 bpm (p<0.0.001)).

[0332] Hence, the reduction in heart rate induced by doxorubicin was significantly attenuated by the treatment with compounds I-C, I-J and I-K.