NOVEL NICOTINAMIDE DI-NUCLEOTIDE DERIVATIVES AND USE THEREOF

Abstract

A compound of formula I or pharmaceutically acceptable salts and/or solvates thereof:

##STR00001##

Also, compositions including at least one compound of formula I, such a pharmaceutical composition, a food composition, and a cosmetic composition. Further, a method for preparing compounds of formula I and their use as therapeutic compounds for use in the treatment of pain, antineoplastic-induced cardiotoxicity or sickle cell disease.

Claims

1.-17. (canceled)

18. A compound of formula I ##STR00038## or pharmaceutically acceptable salts and/or solvates thereof, for use as a medicament, wherein: X.sub.1 and X.sub.2 are independently selected from O, CH.sub.2, S, Se, CHF, CF.sub.2 and C═CH.sub.2; R.sub.1 and R.sub.13 are independently selected from H, azido, cyano, C1-C8 alkyl, C1-C8 thio-alkyl, C1-C8 heteroalkyl and OR, wherein R is selected from H and C1-C8 alkyl; R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.9, R.sub.10, R.sub.11, R.sub.12 are independently selected from H, halogen, azido, cyano, hydroxyl, C1-C12 alkyl, C1-C12 thio-alkyl, C1-C12 heteroalkyl, C1-C12 haloalkyl and OR; wherein R is selected from H, C1-C12 alkyl, C(O)(C1-C12)alkyl, C(O)NH(C1-C12)alkyl, C(O)O(C1-C12)alkyl, C(O)aryl, C(O)(C1-C12)alkyl aryl, C(O)NH(C1-C12)alkyl aryl, C(O)O(C1-C12)alkyl aryl or C(O)CHR.sub.AANH.sub.2, wherein R.sub.AA is a side chain selected from a proteinogenic amino acid; R.sub.6 and R.sub.8 are independently selected from H, azido, cyano, C1-C8 alkyl and OR; wherein R is selected from H and C1-C8 alkyl; R.sub.7 and R.sub.14 are independently selected from H, OR, NHR, NRR′, NH—NHR, SH, CN, N.sub.3 and halogen; wherein R and R′ are each independently selected from H, C1-C8 alkyl, (C1-C8)alkyl aryl; Y.sub.1 and Y.sub.2 are independently selected from CH, CH.sub.2, C(CH.sub.3).sub.2 or CCH.sub.3; M is selected from H or a suitable counterion; represents a single or a double bound depending on Y.sub.1 and Y.sub.2; and represents the alpha or beta anomer depending on the position of R.sub.1 and R.sub.13.

19. The compound for use according to claim 18, wherein X.sub.1 and X.sub.2 each independently represents an oxygen.

20. The compound for use according to claim 18, wherein R.sub.1 and/or R.sub.13 each independently represents a hydrogen.

21. The compound for use according to claim 18, wherein R.sub.6 and/or R.sub.8 each independently represents a hydrogen.

22. The compound for use according to claim 18, wherein R.sub.3, R.sub.4, R.sub.10, R.sub.11 are identical and represent each a hydrogen.

23. The compound for use according to claim 18, wherein R.sub.2, R.sub.5, R.sub.9 and R.sub.12 are identical and represent each a hydroxyl.

24. The compound for use according to claim 18, wherein Y.sub.1 and Y.sub.2 each independently represents a CH.

25. The compound for use according to claim 18, wherein Y.sub.1 and Y.sub.2 each independently represents a CH.sub.2.

26. The compound for use according to claim 18, selected from compounds of formula I-A to I-F: ##STR00039##

27. The compound for use according to claim 18, The compound for use according to claim 18, wherein the compound is of formula I-A, I-B or I-C.

28. A compound of formula I′ ##STR00040## or pharmaceutically acceptable salts and/or solvates thereof, wherein: X.sub.1 and X.sub.2 are independently selected from O, CH.sub.2, S, Se, CHF, CF.sub.2 and C═CH.sub.2; R.sub.1 and R.sub.13 are independently selected from H, azido, cyano, C1-C8 alkyl, C1-C8 thio-alkyl, C1-C8 heteroalkyl and OR, wherein R is selected from H and C1-C8 alkyl; R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.9, R.sub.10, R.sub.11, R.sub.12 are independently selected from H, halogen, azido, cyano, hydroxyl, C1-C12 alkyl, C1-C12 thio-alkyl, C1-C12 heteroalkyl, C1-C12 haloalkyl and OR; wherein R is selected from H, C1-C12 alkyl, C(O)(C1-C12)alkyl, C(O)NH(C1-C12)alkyl, C(O)O(C1-C12)alkyl, C(O)aryl, C(O)(C1-C12)alkyl aryl, C(O)NH(C1-C12)alkyl aryl, C(O)O(C1-C12)alkyl aryl or C(O)CHR.sub.AANH.sub.2, wherein R.sub.AA is a side chain selected from a proteinogenic amino acid; R.sub.6 and R.sub.8 are independently selected from H, azido, cyano, C1-C8 alkyl and OR; wherein R is selected from H and C1-C8 alkyl; R.sub.7 and R.sub.14 are independently selected from H, OR, NHR, NRR′, NH—NHR, SH, CN, N.sub.3 and halogen; wherein R and R′ are each independently selected from H, C1-C8 alkyl, (C1-C8)alkyl aryl; Y.sub.1 and Y.sub.2 are independently selected from CH, CH.sub.2, C(CH.sub.3).sub.2 or CCH.sub.3; M is selected from H or a suitable counterion; represents a single or a double bound depending on Y.sub.1 and Y.sub.2; and represents the alpha or beta anomer depending on the position of R.sub.1 and R.sub.13, with the proviso that when; X.sub.1 and X.sub.2 are oxygen; R.sub.1, R.sub.3, R.sub.4, R.sub.6, R.sub.5, R.sub.10, R.sub.11, and R.sub.13 are hydrogen; R.sub.2, R.sub.5, R.sub.9 and R.sub.12 are hydroxyl; R.sub.7 and R.sub.14 are NH.sub.2; and Y.sub.1 and Y.sub.2 are independently selected from CH or CH.sub.2, then at least one of represent the alpha anomer.

29. A pharmaceutical composition comprising at least one compound for use according to claim 18, and at least one pharmaceutically acceptable carrier.

30. A method of treatment of pain, antineoplastic-induced cardiotoxicity or sickle cell disease in a subject, comprising administering to a subject in need thereof an effective amount of the compound for use according to claim 18.

31. A food composition comprising at least one compound for use according to claim 18, and at least one acceptable carrier and/or diluent.

32. A cosmetic composition comprising at least one compound for use according to claim 18, and at least one acceptable carrier and/or diluent.

33. A method for preparing a compound of formula I as defined in claim 18 comprising the following steps: 1) mono-phosphorylation of a compound of formula X, ##STR00041## wherein: X.sub.1, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, Y.sub.1, and are as defined above, to give compound of formula XI, ##STR00042## wherein: X.sub.1, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, Y.sub.1, and are as defined above; 2) hydrolysis of compound of formula XI obtained in step 1), to give compound of formula XII ##STR00043## wherein: X.sub.1, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, Y.sub.1, and are as defined above; 3) reacting compound of formula XII obtained in step 2) with compound of formula XIII, ##STR00044## obtained as described in step 1) and wherein: X.sub.2, R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, R.sub.13, R.sub.14, Y.sub.2, and are as defined above, to give compound of formula I.

34. The method according to claim 33, further comprising a step of reducing the compound of formula I or formula I′ obtained in step 3), to give the compound of formula I, wherein Y.sub.1 and Y.sub.2 each independently represents a CH.sub.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0365] FIGS. 1A & C are graphs showing the basal nociceptive threshold for all experimental group.

[0366] FIGS. 1B & D are graphs showing the basal nociceptive scores for all experimental group.

[0367] FIG. 2A is a graph showing the nociceptive threshold for CYP-induced visceral pain at 2 h, 4 h and 6 h in comparison with basal value within the vehicle group. Friedman test or One-way Anova and Two-way RM Anova by both factors with Dunn's or Dunnett's post test vs Basal, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001.

[0368] FIG. 2B is a graph showing the nociceptive scores for CYP-induced visceral pain at 2 h, 4 h and 6 h in comparison with basal value within the vehicle group. Friedman test or One-way Anova and Two-way RM Anova by both factors with Dunn's or Dunnett's post test vs Basal, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001.

[0369] FIG. 3A is a graph showing the nociceptive threshold for the effects of NMN, pro-drug A and pro-drug B on CYP-induced allodynia at 2 h.

[0370] FIG. 3B is a graph showing the nociceptive threshold for the effects of NMN, pro-drug A and pro-drug B on CYP-induced allodynia at 4 h.

[0371] FIG. 3C is a graph showing the evolution of the nociceptive threshold following administration of compounds I-B and I-C over 6 hours. Two-way RM Anova with Sidak's multiple comparison test vs Vehicle group, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001.

[0372] FIG. 4 is a graph showing the effects NMN on CYP-induced visceral pain (nociceptive scores) at 2 h (FIG. 4A) and 4 h (FIG. 4B). Two-way RM Anova, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001

[0373] FIG. 5 is a graph showing the effects pro-drug A on CYP-induced visceral pain (nociceptive scores) at 2 h (FIG. 5A) and 4 h (FIG. 5B). Two-way RM Anova, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001.

[0374] FIG. 6 is a graph showing the effects pro-drug B on CYP-induced visceral pain (nociceptive scores) at 2 h (FIG. 6A) and 4 h (FIG. 6B). Two-way RM Anova, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001.

[0375] FIG. 7 is a graph showing the effects compound I-B on CYP-induced visceral pain (nociceptive scores). at 2 h (FIG. 7A), 4 h (FIG. 7B) and 6 h (FIG. 7C). Two-way RM Anova, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001.

[0376] FIG. 8 is a graph showing the effects compound I-C on CYP-induced visceral pain (nociceptive scores). at 2 h (FIG. 8A), 4 h (FIG. 8B) and 6 h (FIG. 8C). Two-way RM Anova, .sup.#p<0.05, .sup.##p<0.01, .sup.###p<0.001, .sup.####p<0.0001.

[0377] FIG. 9 is a histogram showing the survival rate of mice 5 days after DOX (20 mg/kg) or vehicle induction, with and without treatments. ##p<0.01: Fisher's test Dox mice treated with vehicle vs control mice, £p<0.05, ££p<0.01: Fisher's test Dox mice treated with vehicle vs Dox mice treated with NMN analogs.

[0378] FIG. 10A shows the body weight evolution of mice treated with NMN, compounds I-B and I-C (180 mg/kg) or vehicle, before (light gray symbol) and 5 days after saline solution or DOX (20 mg/kg) injection (dark gray symbol). £££p<0.001: Two-way ANOVA followed by Bonferroni post-test body weight before Dox injection vs 5 days after Dox injection.

[0379] FIG. 10B is a histogram showing the bodyweight gain calculated as follow: BW at the day of sacrifice minus BW before injection of mice treated with NMN, compounds I-B and I-C (180 mg/kg) or vehicle, with and without DOX (20 mg/kg) injection. ***p<0.001: Mann-Whitney test Dox mice treated with vehicle vs control mice, $$p<0.01, $$$p<0.001 One-way ANOVA followed by post-hoc Dunnett test Dox mice treated with vehicle n vs Dox mice treated with NMN analogs.

[0380] FIG. 11 is a histogram showing left ventricle (LV) end-diastolic (FIG. 11A) and end-systolic volumes (FIG. 11B), and ejection fraction (FIG. 11C) 5 days after saline solution or DOX (20 mg/kg) injection. **p<0.01, ***p<0.001: Mann-Whitney test Dox mice treated with vehicle vs control mice, $p<0.05, $$$p<0.001 Kruskal-Wallis test followed by post-hoc Dunn test Dox mice treated with vehicle vs Dox mice treated with NMN analogs.

[0381] FIG. 12 is a histogram showing LV end diastolic and end systolic diameters (FIGS. 12A and 12B respectively), fractional shortening (FIG. 12C) and heart rate (FIG. 12D) 5 days after saline solution or DOX (20 mg/kg) injection. **p<0.01, ***p<0.001: t-test or Mann-Whitney test Dox mice treated with vehicle vs control mice, $$$p<0.001: One-way ANOVA followed by post-hoc Dunnett test or Kruskal-Wallis test followed by post-hoc Dunn Dox mice treated with vehicle vs Dox mice treated with NMN analogs (180 mg/kg) or vehicle.

[0382] FIG. 13 is a histogram showing LV anterior wall thickness in systole and in diastole (FIGS. 13A and 13B respectively) and posterior wall thickness in systole and in diastole (FIGS. 13C and 13D respectively) 5 days after saline solution or DOX (20 mg/kg) injection. *p<0.05, **p<0.01: Mann-Whitney test Dox mice treated with vehicle vs control mice.

[0383] FIG. 14 is a histogram graph showing heart weight (FIG. 14A) and heart weight normalized to tibial length (FIG. 14B) 5 days after saline solution or DOX (20 mg/kg) injection. ***p<0.001: t-test Dox mice treated with vehicle vs control mice.

[0384] FIG. 15 is a histogram graph showing LDH concentrations (U/L, FIG. 15A) and LDH (fold change, FIG. 15B) in the plasma of mice 5 days after saline solution or DOX (20 mg/kg) injection. **p<0.01: Mann-Whitney test Dox mice treated with vehicle vs control mice; $p<0.05: Kruskal-Wallis test Dox mice treated with vehicle vs Dox mice treated with NMN analogs (180 mg/kg) or vehicle.

[0385] FIG. 16 is a histogram graph showing the ability of NMN to prevent sickling of SS RBCs at a 1% O.sub.2. Non-parametric one-way ANOVA followed by Kruskal-Wallis test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0386] FIG. 17 is a histogram graph showing the ability of compounds I-B to prevent sickling of SS RBCs at a 1% O.sub.2. Non-parametric one-way ANOVA followed by Kruskal-Wallis test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0387] FIG. 18 is a histogram graph showing the ability of compounds I-C to prevent sickling of SS RBCs at a 1% O.sub.2. Non-parametric one-way ANOVA followed by Kruskal-Wallis test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

EXAMPLES

[0388] The present invention is further illustrated by the following examples.

Example 1: Synthesis of Compounds of the Invention

Material and Methods

[0389] All materials were obtained from commercial suppliers and used without further purification. Thin-layer chromatography was performed on TLC plastic sheets of silica gel 60F254 (layer thickness 0.2 mm) from Merck. Column chromatography purification was carried out on silica gel 60 (70-230 mesh ASTM, Merck). Melting points were determined either on a digital melting point apparatus (Electrothermal IA 8103) and are uncorrected or on a Kofler bench type WME (Wagner & Munz). IR, .sup.1H, .sup.19F and .sup.13C NMR spectra confirmed the structures of all compounds. IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer and 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 .sup.1H, 75 or 100 MHz for .sup.13C and 282 or 377 MHz for .sup.19F spectra. Chemical shifts (δ) were expressed in parts per million relative 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. Chemical shifts are given in ppm and peak multiplicities are designated as follows: s, singlet; br s, broad 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 Nationale de la Recherche Scientifique) and were recorded on a Waters spectrometer using electrospray ionization-TOF (ESI-TOF).

General Experimental Procedures

Step 1: Synthesis of Compound of Formula X-1

[0390] Compound of formula XIV (1.0 equiv.) is dissolved in dichloromethane. Nicotinamide of formula XV (1.50 equiv.) and TMSOTf (1.55 equiv.) are added at room temperature. The reaction mixture is heated to reflux and stirred until completion is reached. The mixture is cooled down to room temperature and filtered. The filtrate is concentrated to dryness to give the crude NR tetraacetate of formula X-1.

Step 2: Synthesis of Compound of Formula X

[0391] The crude NR tetraacetate of formula X-1 is dissolved in methanol and cooled down to −10° C. 4.6 M Ammonia in methanol (3.0 equiv.) is added at −10° C. and the mixture is stirred at this temperature until completion is reached. Dowex HCR (H+) is added until pH=6-7. The reaction mixture is warmed 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 crude NR triflate of formula X.

Step 3: Synthesis of Compound of Formula XI

[0392] The solution of crude NR triflate in acetonitrile is diluted with trimethyl phosphate (10.0 equiv.). Acetonitrile is distilled under vacuum and the mixture is cooled to −10° C. Phosphorus oxychloride (4.0 equiv.) is added at −10° C. and the mixture is stirred at −10° C. until completion.

Step 4 and Step 5: Synthesis of Compound of Formula I-A

[0393] The mixture is hydrolyzed by addition of a 50/50 mixture of acetonitrile and water, followed by addition of tert-butyl methyl ether. The mixture is filtered and the solid is dissolved in water. The aqueous solution is neutralized by addition of sodium bicarbonate and extracted with dichloromethane. The aqueous layer is concentrated to dryness to give a crude mixture of NMN and di-NMN of formula I-A.

Isolation of Di-NMN of Formula I-A:

[0394] NMN and di-NMN of formula I-A are separated by purification on Dowex 50wx8 with water elution. The fractions containing di-NMN are concentrated to dryness. The residue is purified by column chromatography on silica gel (gradient isopropanol/water). Pure fractions are combined and concentrated. The residue is freeze-dried to afford di-NMN as a beige solid.

[0395] .sup.31P RMN: δ (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm dans D.sub.2O)=−11.72; .sup.1H RMN: δ (ppm, reference TMS: 0 ppm dans D.sub.2O)=4.20 (ddd, J.sub.H-H=11.9, 3.5, 2.4 Hz, 2H), 4.35 (ddd, J.sub.H-H=11.9, 3.9, 2.2 Hz, 2H), 4.43 (dd, J.sub.H-H=5.0, 2.6 Hz, 2H), 4.53 (t, J.sub.H-H=5.0 Hz, 2H), 4.59 (m, 2H), 6.16 (d, J.sub.H-H=5.4 Hz, 2H), 8.26 (dd, J.sub.H-H=8.1, 6.3 Hz, 2H), 8.93 (d, J.sub.H-H=8.1 Hz, 2H), 9.25 (d, J.sub.H-H=6.2 Hz, 2H), 9.41 (s, 2H); .sup.13C RMN: δ (ppm, reference TMS: 0 ppm dans D.sub.2O)=64.84 (CH.sub.2), 70.73 (CH), 77.52 (CH), 87.11 (CH), 99.88 (CH), 128.65 (CH), 133.89 (Cq), 139.84 (CH), 142.54 (CH), 146.04 (CH), 165.64 (Cq); MS (ES+): m/z=122.8 [Mnicotinamide+H]+, 650.8 [M+H]+.

Synthesis of Compound of Formula I-B Phosphorus oxychloride (3.0 eq.) is added to trimethylphosphate (20.0 eq.) at −5° C. β-NR chloride (1.0 eq.) is added by portions at −5° C. and the reaction mixture stirred overnight at −5° C. Morpholine (3.0 eq.) is added dropwise at −10/0° C. and the mixture stirred for 2-3 h. α-NMN (1.0 eq.) is then added by portions at −5° C. and the reaction mixture stirred at −5° C. overnight. Hydrolysis is performed by dropwise addition of water (5 vol.) at −10/0° C. and the mixture is stirred until complete homogeneization at 10-15° C. The reaction mixture is then extracted with dichloromethane (6*10 vol.) and the aqueous phase neutralized by eluting through Purolite A600E formate form resin (theoretical amount to neutralize HCl coming from POCl.sub.3). The eluate is then concentrated on vacuum at 45/50° C. to give the crude containing the α,β-diNMN of formula I-B. Elution with water through Dowex 50wx8 100-200 mesh H.sup.+ form resin allows removing of some impurities. Fractions containing compound I-B are combined and concentrated on vacuum at 45-50° C. The crude is then purified by preparative chromatography on Luna Polar RP 10 μm stationary phase with elution with a 10 mM NaH.sub.2PO.sub.4 aqueous solution. Pure fractions are combined and eluted with water on Purolite C100EH H.sup.+ form resin (needed quantity to fully exchange Na.sup.+ by H.sup.+), then eluted on Purolite A600E acetate form resin (needed quantity to fully exchange H.sub.2PO.sub.4.sup.− by acetate). The eluate is concentrated on vacuum and the residue freeze-dried to afford compound I-B as a white solid.

[0396] .sup.31P RMN: δ (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm dans D.sub.2O)=−11.87, −11.69, −11.46, −11.29; .sup.1H RMN: δ (ppm, reference TMS: 0 ppm dans D.sub.2O)=4.10 (ddd, J=11.1, 6.1, 3.1 Hz, 1H), 4.15-4.25 (m, 2H), 4.36 (ddd, J=12.2, 4.4, 2.4 Hz, 1H), 4.40 (dd, J=4.9, 2.4 Hz, 1H), 4.44 (dd, J=5.0, 2.7 Hz, 1H), 4.53 (t, J=5.0 Hz, 1H), 4.5 (m, 1H), 4.85 (m, 1H), 4.92 (t, J=5.3 Hz, 1H), 6.15 (d, J=5.5 Hz, 1H), 6.51 (d, J=5.7 Hz, 1H), 8.14 (dd, J=8.0, 6.3 Hz, 1H), 8.26 (dd, J=8.1, 6.3 Hz, 1H), 8.88 (d, J=8.1 Hz, 1H), 8.92 (d, J=8.1 Hz, 1H), 9.02 (d, J=6.3 Hz, 1H), 9.24 (s, 1H), 9.26 (d, J=6.4 Hz, 1H), 9.40 (s, 1H); .sup.13C RMN: δ (ppm, reference TMS: 0 ppm dans D.sub.2O)=64.83, 64.87 (CH2), 65.30, 65.35 (CH2), 70.65 (CH), 70.74 (CH), 71.92 (CH), 77.51 (CH), 87.03, 87.10 (CH), 87.19, 87.26 (CH), 96.57 (CH), 99.83 (CH), 126.89 (CH), 128.54 (CH), 132.44 (Cq), 133.81 (Cq), 139.85 (CH), 140.92 (CH), 142.50 (CH), 143.49 (CH), 145.06 (CH), 145.97 (CH), 165.64 (Cq), 165.88 (Cq); MS (ES+): m/z=122.8 [Mnicotinamide+H]+, 650.9 [M+H]+.

Synthesis of Compound of Formula I-C

[0397] Phosphorus oxychloride (3.0 eq.) is added to trimethylphosphate (20.0 eq.) at −5° C. α-NR chloride (1.0 eq.) is added by portions at −5° C. and the reaction mixture stirred overnight at −5° C. Morpholine (3.0 eq.) is added dropwise at −10/0° C. and the mixture stirred for 2-3 h. α-NMN (1.0 eq.) is then added by portions at −5° C. and the reaction mixture stirred at −5° C. overnight. Hydrolysis is performed by dropwise addition of water (5 vol.) at −10/0° C. and the mixture is stirred until complete homogenization at 10-15° C. The reaction mixture is then extracted with dichloromethane (6*10 vol.) and the aqueous phase neutralized by eluting through Purolite A600E formate form resin (theoretical amount to neutralize HCl coming from POCl.sub.3). The eluate is then concentrated on vacuum at 45/50° C. to give the crude containing the α,α-diNMN of formula I-C. Elution with water through Dowex 50wx8 100-200 mesh H.sup.+ form resin allows removing of some impurities. Fractions containing the compound I-C are combined and concentrated on vacuum at 45-50° C. The crude is then purified by preparative chromatography on Luna Polar RP 10 μm stationary phase with elution with a 10 mM NaH.sub.2PO.sub.4 aqueous solution. Pure fractions are combined and eluted with water on Purolite C100EH H.sup.+ form resin (needed quantity to fully exchange Na.sup.+by H.sup.+), then eluted on Purolite A600E acetate form resin (needed quantity to fully exchange H.sub.2PO.sub.4.sup.− by acetate). The eluate is concentrated on vacuum and the residue freeze-dried to afford compound I-C as a white solid.

[0398] .sup.31P RMN: δ (ppm, reference 85% H.sub.3PO.sub.4: 0 ppm dans D.sub.2O)=−11.40; .sup.1H RMN: δ (ppm, reference TMS: 0 ppm dans D.sub.2O)=4.14 (ddd, J=11.4, 3.4, 2.8 Hz, 2H), 4.23 (ddd, J=11.6, 3.3, 2.8 Hz, 2H), 4.44 (dd, J=4.8, 2.3 Hz, 2H), 4.88 (m, 2H), 4.96 (t, J=5.3 Hz, 2H), 6.54 (d, J=5.7 Hz, 2H), 8.15 (dd, J=8.1, 6.2 Hz, 2H), 8.89 (d, J=8.1 Hz, 2H), 9.05 (d, J=6.3 Hz, 2H), 9.26 (s, 2H); .sup.13C RMN: δ (ppm, reference TMS: 0 ppm dans D.sub.2O)=65.37 (CH2), 70.70 (CH), 71.95 (CH), 87.30 (CH), 96.62 (CH), 126.91 (CH), 132.45 (Cq), 140.94 (CH), 143.52 (CH), 145.07 (CH), 165.90 (Cq); MS (ES+): m/z=122.7[Mnicotinamide+H]+, 650.8 [M+H]+.

Example 2: Evaluation of Compounds of the Invention in the Acute Cyclophosphamide (CYP)-Induced Cystitis Model in Female Sprague-Dawley Rats

[0399] The aim of the present study was to evaluate, the effects of oral administration of Nicotinamide Mono-Nucleoside (NMN), Pro-drug A (alpha-NMN) and Pro-drug B (compound I-A), compounds I-B and I-C at 500 mg/kg on visceral pain response in the acute cyclophosphamide (CYP)-induced cystitis model in female Sprague-Dawley rats.

I. Materials and Methods

Animals

[0400] Female Sprague-Dawley rats, 7 weeks at delivery

Pharmacological Treatment

[0401] NMN: 500 mg/kg [0402] Pro-drug A: 500 mg/kg [0403] Pro-drug B (compound I-A): 500 mg/kg [0404] Compound I-B: 500 mg/kg [0405] Compound I-C; 500 mg/kg [0406] Vehicle: distilled water [0407] Route of administration: per os (p.o.), 5 mL/kg [0408] Frequency of administration: once at DO, 15 min prior CYP intraperitoneal (i.p.) injection.

CYP-Induced Acute Cystitis

[0409] CYP was injected i.p. at 150 mg/kg in a final volume of 5 mL/kg in saline.

Mechanical Stimulation Using Von Frey Filaments

[0410] Rats were placed in individual Plexiglas boxes with a wire mesh floor and allowed to adapt to the chamber for at least 30 min before any test starts. [0411] 8 von Frey filaments with increasing forces of 1, 2, 4, 6, 8, 10, 15 and 26 g were used. [0412] Each calibrated filament was applied 3 times in the lower abdominal area close to the urinary bladder.

Nociceptive Behaviors Scoring for Each Application

[0413] Score 0=no response [0414] Score 1=retraction of the abdomen [0415] Score 2=trampling or change of position [0416] Score 3=flinching or abdominal curvature or licking of the site stimulated with von Frey filaments
For Each Rat, Results were Expressed as: [0417] Nociceptive threshold: first von Frey force for which the stimulus is perceived as painful (score ≥1 is obtained)

[0418] =>lowered threshold=allodynia [0419] Nociceptive score: % of the maximal response (total=9 for 3 pooled applications) for each filament

[0420] =>Global pain response

Experimental Group:

[0421]

TABLE-US-00002 TABLE 2 Group Treatment Injection n 1 Vehicle (5 mL/kg) CYP 6 2 NMN (500 mg/kg) CYP 6 3 Pro-drug A (500 mg/kg) CYP 6 4 Pro-drug B (500 mg/kg) CYP 6 5 Compound I-B (500 mg/kg) CYP 6 6 Compound I-C (500 mg/kg) CYP 6

II. Results and Discussion

[0422] 1. Basal Nociceptive Parameters (Before CYP Injection) for all Experimental Groups

[0423] The results show (FIGS. 1A, 1B, 1C and 1D) that basal nociceptive responses were similar between all experimental groups (before CYP injection).

[0424] 2. CYP-Induced Visceral Pain at 2 h and 4 h Post-Injection (in Comparison with Basal Value within the Vehicle Group)

[0425] The results show that in comparison to basal response, CYP (150 mg/kg, i.p.) induced a significant decrease in nociceptive threshold (FIG. 2A) and a significant increase in nociceptive scores (FIG. 2B) at 2 h, 4 h and 6 h.

[0426] 3. Effect of NMN, Pro-Drug a, Pro-Drug B, Compound I-B and Compound I-C on CYP-Induced Allodynia (Nociceptive Threshold)

[0427] The results show that in comparison to vehicle: [0428] NMN (500 mg/kg, p.o) led to a slight increase in nociceptive threshold at +2 h (FIG. 3A) and +4 h (FIG. 3B) with an effect just above the margin of statistical significance at +4 h (p=0.063), [0429] Pro-Drug A (500 mg/kg, p.o) led to a non-significant increase in nociceptive threshold at +2 h (FIG. 3A) and a significant increase at +4 h (FIG. 3B), [0430] Pro-Drug B (500 mg/kg, p.o) led to a significant increase in nociceptive threshold at +4 h (FIG. 3B). [0431] Compound I-B (500 mg/kg, p.o) led to a significant increase in nociceptive threshold at +6 h following CYP induction (FIG. 3C). [0432] Compound I-C (500 mg/kg, p.o) led to a significant increase in nociceptive threshold at +6 h following CYP induction (FIG. 3C).

[0433] 4. Effects of NMN, Pro-Drug A and Pro-Drug B, Compounds I-B and I-C on CYP-Induced Visceral Pain (Nociceptive Scores)

[0434] The results show that in comparison to vehicle: [0435] NMN (500 mg/kg, p.o) led to a significant decrease in nociceptive scores at +2 (FIG. 4A) and +4 h (FIG. 4B), [0436] Pro-Drug A (500 mg/kg, p.o) led to a decrease in nociceptive scores at +2 (FIG. 5A) and +4 h (FIG. 5B) that achieved the statistical level only at +4 h, [0437] Pro-Drug B (500 mg/kg, p.o) led to a significant decrease in nociceptive scores at +4 h (FIG. 6B) (no effect was observed at +2 h (FIG. 6A)), [0438] Compound I-B (500 mg/kg, p.o) led to a significant decrease in nociceptive scores at +2 (FIG. 7A), +4 h (FIG. 7B) and +6 h (FIG. 7C), [0439] Compound I-C (500 mg/kg, p.o) led to a significant decrease in nociceptive scores at +2 (FIG. 8A), +4 h (FIG. 8B) and +6 h (FIG. 8C).

[0440] 5. Summary of Results

[0441] Basal nociceptive responses were similar between all experimental groups (before CYP injection).

[0442] In comparison to basal response, effects of CYP (150 mg/kg, i.p.) at 2 and 4 hours were characterized by: [0443] A significant decrease in nociceptive threshold at +2, +4 h and +6 h, [0444] A significant increase in nociceptive scores at +2, +4 h and +6 h.

[0445] In comparison to vehicle, in CYP-injected rats, effects of NMN (500 mg/kg, p.o.) led to: [0446] A slight increase in nociceptive threshold at +2 and +4 h with an effect just above the margin of statistical significance at +4 h (p=0.063), [0447] A significant decrease in nociceptive scores at +2 and +4 h.

[0448] In comparison to vehicle, in CYP-injected rats, effects of Pro-drug A (500 mg/kg, p.o.) were characterized by: [0449] An increase in nociceptive threshold at +2 and +4 h with an effect that reached significance at +4 h, [0450] A decrease in nociceptive scores at +2 and +4 h that achieved significance at +4 h.

[0451] In comparison to vehicle, in CYP-injected rats, effects of Pro-drug B (500 mg/kg, p.o.) led to: [0452] A significant increase in nociceptive threshold at +4 h, [0453] A significant decrease in nociceptive scores at +4 h.

[0454] In comparison to vehicle, in CYP-injected rats, effects of compound I-B (500 mg/kg, p.o.) led to: [0455] A significant decrease in nociceptive scores at +2, +4 h and +6 h. [0456] A significant increase in nociceptive thresholds for 6 hours following CYP injection.

[0457] In comparison to vehicle, in CYP-injected rats, effects of compound I-C (500 mg/kg, p.o.) led to: [0458] A significant decrease in nociceptive scores at +2, +4 h and +6 h, [0459] A significant increase in nociceptive thresholds for 6 hours following CYP injection.

III. Conclusion

[0460] A single intraperitoneal injection of CYP (150 mg/kg) induced visceral pain at 2-, 4- and 6-hours post-injection, thus validating the model.

[0461] Single oral treatment of NMN (500 mg/kg) alleviated CYP-induced visceral pain at both evaluated time points (+2 h and +4 h) with a higher level of significance at +4 h.

[0462] Pro-drug A (500 mg/kg, p.o.) reduced CYP-induced visceral pain at both evaluated time points (+2 h and +4 h) with significance at +4 h.

[0463] In CYP-injected rats, oral treatment with Pro-drug B (compound I-A) (500 mg/kg, p.o.) resulted in significant anti-nociceptive activity at +4 h.

[0464] In CYP-injected rats, oral treatment with compounds I-B and I-C (500 mg/kg, p.o.) displayed significant anti-nociceptive activity at the three evaluated time points, +2 h, +4 h and +6 h.

Example 3: Evaluation of Compounds of the Invention in a Model of Doxorubicin-Induced Cardiotoxicity

[0465] The aim of the present study was to evaluate, the effects of i.p administration of Nicotinamide Mononucleotide (NMN), compound I-B and compound I-C at 180 mg/kg in the progression of a cardiotoxicity induced by doxorubicin.

I. Materials and Methods

Material

Animals:

[0466] 76 male mice, 8-week-old at the arrival were obtained from Janvier Labs, Le Genest St Isle, 53941 St Berthevin, France. Each animal was identified with electronic chip. Each cage was numbered. Based on the animal number/cage and number of cages, the animals were assigned of unique number with the name of group and mice number.

[0467] The matching cards that were used to identify cages where experimental animals were housed contained the following information: the name of the experiment, the number of the experiment and the cage number.

Methods

[0468] 1. Preparation of Formulation:

[0469] The powder of NMN, compounds I-B and I-C (180 mg/kg) were dissolved in vehicle (the solution is used at room temperature for maximum 1 day). A fresh sample for each administration was prepared every day except the week-end (the solution is prepared on Saturday and is used on Saturday and Sunday).

[0470] 2. Doxororubicin-induced cardiotoxicity

[0471] Cardiotoxicity was induced by a single intraperitoneal injection of doxorubicin (DOX) at 20 mg/kg. Doxorubicin was prepared at 2 mg/mL and volume of administration was 10 mL/Kg.

[0472] Mortality rate was followed-up all along the experimental phase.

[0473] 3. Experimental Groups

[0474] Group description:

[0475] Group 1: Vehicle (i.p.)

[0476] Group 2: Doxorubicin (20 mg/kg)

[0477] Group 3: Doxorubicin (20 mg/kg)+test compound 180 mg/kg (NMN)

[0478] Group 4: Doxorubicin (20 mg/kg)+test compound 180 mg/kg (compound I-B)

[0479] Group 5: Doxorubicin (20 mg/kg)+test compound 180 mg/kg (compound I-C)

[0480] Group repartition:

[0481] Each group involved 14-24 mice.

[0482] As set forth in the regulations for Non-clinical Laboratory Studies, test and control animal groups were maintained under identical conditions. The intended duration of study was 11 days.

[0483] 4. Induction with Doxorubicin

[0484] At D0, mice were injected with DOX (20 mg/kg) by intraperitoneal route.

[0485] 5. Treatment

[0486] The treatment with NMN, compounds I-B and I-C was initiated from 5 days before DOX injection, once per day, from D-5 to DO.

[0487] Mice were i.p treated with NMN, compounds I-B and I-C 30 min before DOX injection.

[0488] Mice were i.p treated with NMN, compounds I-B and I-C for the duration of the experiment (D0 to D5) once per day. Last injection occurred 24 hours before sacrifice.

[0489] 6. Body Weight, Survival Rate and Clinical Examination

[0490] The bodyweight was assessed at inclusion and at D5.

[0491] The survival rate was recorded every day until the end of the experiment (D5).

[0492] 7. Blood Collection

[0493] Retro-orbital blood collection was performed at the inclusion and at 1 and 5 days after the infection with DOX to assess biomarker (LDH and creatinine).

[0494] 8. Organs Collection

[0495] At D5, heart, and tibia were collected.

[0496] 9. Assessment of Cardiac Function by Echocardiography

[0497] Echocardiography (ECG) was performed 5 days after doxorubicin injection in anesthetized (isoflurane 1.5-2%) animals with non-invasive two-dimensional echocardiography (VF16-5 probe, Siemens, Acuson NX3 Elite). After removing hairs on the chest, numeric images of the heart were obtained in both parasternal long-axis and short axis views.

[0498] The following cardiac function were assessed during ECG: [0499] Left ventricle (LV) end-systolic and end-diastolic inner diameter; [0500] LV end-systolic and end-diastolic volume, [0501] Fractionnal shortening; [0502] Ejection fraction; [0503] Heart Rate; and [0504] Anterior and posterior wall thickness in Diastole and in systole.

II. Results and Discussion

[0505] 1. Survival Rate

[0506] FIG. 9 shows the percentage of survival of mice induced or not with DOX (20 mg/kg), 5 days after doxorubicin injection.

[0507] DOX mice were treated with NMN, compounds I-B and I-C (180 mg/kg) or vehicle.

[0508] As shown in FIG. 9, almost 50% of doxorubicin mice treated with vehicle died before the end of experimental protocol.

[0509] Treatment with NMN tended to improve the survival rate (78% of survival) without reaching statistical significance. However, treatment with compounds I-B or I-C significantly improved survival rate (98% and 100% of survival respectively) compared to untreated groups (50% of survival).

[0510] 2. Body Weight

[0511] FIG. 10A shows the body weight evolution of mice treated with NMN, compounds I-B and I-C (180 mg/kg) or vehicle, before (light gray symbol) and 5 days after saline solution or DOX (20 mg/kg) injection (dark gray symbol).

[0512] FIG. 10B shows the body weight gain calculated as follow: bodyweight at the day of sacrifice minus bodyweight before injection.

[0513] Surviving vehicle-treated mice showed major signs of suffering associated with a strong decrease in body weight (−4.2±0.5 g). The body weight loss observed after doxorubicin administration was significantly decreased by NMN, compounds I-B and I-C (p<0.01, p<0.001, p<0.001 respectively).

[0514] 3. Cardiac Function

[0515] 3.1. Left Ventricle End Diastolic/Systolic Volumes and Ejection Fraction

[0516] FIG. 11 shows Left ventricle (LV) end diastolic (FIG. 11A), end systolic volumes (FIG. 11B) and ejection fraction (FIG. 11C) 5 days after saline solution or DOX (20 mg/kg) injection, with and without treatment with NMN, compound I-B and compound I-C.

[0517] As shown in FIG. 11B, doxorubicin induced an significant increase in end-systolic LV (left ventricular) volume without difference in end-diastolic (FIG. 11A) when compared to control group leading to a large decrease in ejection fraction (38.9±1.3% in doxorubicin vehicle group vs 64.8±0.6% in control mice) (FIG. 11C).

[0518] Compared to untreated DOX animals, NMN, compounds I-B and I-C reduced end-systolic LV (FIG. 11B) volume when compared to doxorubicin vehicle group, with statistical significance observed for compound I-B (p<0.05).

[0519] Compared to DOX-induced animals that received vehicle, ejection fraction was significantly improved after treatment with NMN. compounds I-B and I-C (56.9±0.6% in doxorubicin mice treated with NMN (p<0.05). 58.2±0.5% in doxorubicin mice treated with compound I-C (p<0.001) and 60.0±0.6% in doxorubicin mice treated with compound I-B (p<0.001)) (FIG. 11C).

[0520] 3.2. Left Ventricle End Diastolic/Systolic Diameters, Fractional Shortening and Heart Rate

[0521] FIG. 12 shows LV end-diastolic and end-systolic diameters (FIGS. 12A and 12B respectively), fractional shortening (FIG. 12C) and heart rate (FIG. 12D) 5 days after saline solution or DOX (20 mg/kg) injection.

[0522] As shown, in doxorubicin-treated mice, LV internal diameter was significantly increased in systole (FIG. 12B) without significative difference in diastole (FIG. 12A) resulting in a decrease in fractional shortening (33.5±0.4% vs 43.2±0.5% in control mice) (FIG. 12C). Treatment with NMN. compounds I-B and I-C significantly improved fractional shortening to around 38% (p<0.001 for the three groups).

[0523] Moreover. doxorubicin significantly reduced the heart rate (FIG. 12D) when compared to control mice (365.1±23.9 bpm vs 525.6±19.8 bpm respectively). Treatments with NMN. compound I-B and compound I-C resulted in increased heart rates. with compound I-B significantly improving this parameter (470.1±18.8 bpm (p<0.001)).

[0524] 3.3. Left Ventricle Anterior and Posterior Wall Thickness in Systole and in Diastole

[0525] FIG. 13 shows LV anterior wall thickness in systole and in diastole (FIGS. 13A and 13B respectively) and posterior wall thickness in systole and in diastole (FIGS. 13C and 13D respectively) 5 days after saline solution or DOX (20 mg/kg) injection.

[0526] Doxorubicin significantly decreased anterior and posterior wall thickness in systole but not in diastole, and any of treatments had significant effect.

[0527] Treatments with compound NMN, compounds I-B and I-C (180 mg/kg) in DOX mice resulted in non-significant increases of anterior and posterior wall thickness in systole.

[0528] 4. Heart Weight

[0529] FIG. 14 shows Heart weight (FIG. 14A) and heart weight normalized to tibial length (FIG. 14B) 5 days after saline solution or DOX (20 mg/kg) injection.

[0530] DOX mice were treated with compound NMN, compounds I-B and I-C (180 mg/kg) or vehicle.

[0531] As shown in FIGS. 14A and 14B, doxorubicin significantly decreased heart weight when compared to control mice (102.3±4.6 mg vs 128.9±3.3 mg respectively). Treatments with compounds I-B and I-C tended to increase heart weight without reaching significance vs DOX vehicle mice. Similar results were obtained when heart weight was normalized to tibia length.

[0532] 5. Biomarker Assessment

[0533] FIG. 15 shows LDH concentrations (U/L, FIG. 15A) and LDH (fold change, FIG. 15B) in the plasma of mice 5 days after saline solution or DOX (20 mg/kg) injection.

[0534] DOX mice were treated with compound NMN, compounds I-B and I-C (180 mg/kg) or vehicle.

[0535] Plasmatic LDH (lactate deshydrogenase) were measured 5 days after doxorubicin injection. As shown in FIGS. 15A and 15B, doxorubicin induced a 3-fold increase in LDH release compared to control group. Treatment with NMN decreased LDH release by more than 35% without reaching statistical significance. However, treatment with both compounds I-B and I-C resulted in a significant reduction in LDH levels by 50-55% (p<0.05).

[0536] III. Conclusion

[0537] Altogether, results showed that doxorubicin induced cardiac dysfunction characterized by impaired cardiac contractility and cardiac filling, as well as cell cardiac damage. Doxorubicin also led to high mortality, and a strong body weight loss.

[0538] NMN, compounds I-B and I-C treatments significantly improved survival rate, body weight loss and prevented cardiac function degradation as shown by the effects of treatments on ejection fraction, fractional shortening and heart rate.

Example 4: Evaluation of Compounds of the Invention on Sickle Cell Disease Experimental Models

[0539] The aim of the present study was to evaluate, the effects of i.p. administration of Nicotinamide Mononucleotide (NMN), compound I-B and compound I-C at 185 mg/kg as modulator of red blood cell sickling and their potential role in therapy for sickle cell disease on a mouse model of SCD.

[0540] I. Materials and Methods

[0541] Material

[0542] Animals:

[0543] Townes S/S mice on a 129/B6 mixed genetic background.

[0544] Methods

[0545] 1. Preparation of Formulation:

[0546] The powder of NMN, compounds I-B and I-C (185 mg/kg) were dissolved in vehicle (the solution was used at room temperature for maximum 1 day). A fresh sample for each administration was prepared every day except the week-end (the solution is prepared on Saturday and is used on Saturday and Sunday).

[0547] 2. Sickle Red Blood Cell

[0548] In Townes S/S mice, mouse alpha- and beta-globin gene loci are deleted and replaced by human alpha- and beta-globins. When carrying two copies of the beta S allele, mice develop a human sickle cell disease phenotype with sickle-shaped red blood cells seen in blood smears.

[0549] 3. Experimental Groups

[0550] Group description:

[0551] Group I: Vehicle (i.p.)

[0552] Group II: NMN 185 mg/kg

[0553] Group III: compound I-B 185 mg/kg

[0554] Group IV compound I-C 185 mg/kg

[0555] 4. Treatment

[0556] Mice were i.p treated with NMN, compounds I-B and I-C during all the experiment (D0 to D15) once per day. Last injection occurred 24 hours before sacrifice.

[0557] 5. Blood Collection

[0558] Retro-orbital blood collection was performed at the inclusion D0 and at D5, D10 and D15 through facial vein bleeding.

[0559] 6. Ex Vivo

[0560] Red blood cells collected were submitted to hypoxia (1% O.sub.2) for 30 minutes to induce sickling. Percentage of sickle cells was then assessed.

[0561] I. Results and Discussion

[0562] 1. RBC Sickling Under Hypoxia Ex Vivo

[0563] FIGS. 16, 17 and 18 show the ability of NMN (FIG. 16), compounds I-B (FIG. 17) and I-C (FIG. 18) to prevent sickling of SS RBCs at a 1% O.sub.2.

[0564] SS RBCs from treated mice were collected at D0, D5, D10 and D15 and were submitted to hypoxia for 30 minutes in a hypoxic chamber (1% O.sub.2). Percentage of sickling RBCs was then assessed for each time point with compounds NMN, I-B and I-C.

[0565] The results showed that treatment with: [0566] NMN (185 mg/kg, i.p.) led to a significant (p<0.001) decrease of the percentage of sickling cells from 40% at D0 to less than 10% after 15 days treatment of mice (FIG. 16); [0567] Compound I-B (185 mg/kg, i.p.) led to a significant decrease (p<0.0001) of the percentage of sickling cells from 32% at D0 to less than 15% after 15 days treatment of mice (FIG. 17). [0568] Compound I-C (185 mg/kg, i.p.) led to a significant decrease (p<0.001) of the percentage of sickling cells from 31% at D0 to 20% after 15 days treatment of mice (FIG. 18).

[0569] III. Conclusion

[0570] These results indicate that NMN, compounds I-B and I-C prevented SS RBCs sickling under hypoxic conditions.