ANTIMICROBIAL COMPOUNDS

Abstract

The invention relates to phenylene diamine derivatives with certain pharmacological properties resulting from enhancement of epithelial barrier function and/or blocking bacterial translocation through the epithelial barrier. The compounds find use in the treatment of various conditions, including conditions involving translocation of pathogens from the gastrointestinal tract into underlying tissues and vasculature, for example febrile neutropenia, intestinal tissue inflammation, bacteremia and sepsis.

Claims

1. A compound of formula (I) for use in a method of treatment of a disease or condition in an animal, that can be treated by improvement or restoration of epithelial barrier function of the animal, wherein the compound is defined by the following formula: ##STR00034## wherein: Q is selected from Q1, Q2, Q3, Q4, Q5 and Q6: ##STR00035## n is 0 or 1; L is selected from (CH.sub.2).sub.m, C(O), (CH.sub.2).sub.mC(O), O(CH.sub.2).sub.mC(O), OC(O)(CH.sub.2).sub.m(C=O), NHC(O), NRC(O), NH(CH.sub.2).sub.mC(O), NR(CH.sub.2).sub.mC(O), NHC(O)(CH.sub.2).sub.mC(O), NRC(O)(CH.sub.2).sub.mC(O), C(O)NH(CH.sub.2).sub.mC(O), and (CH.sub.2).sub.m(CHR.sup.L)C(O), where m is an integer from 1 to 4; A.sup.1 and A.sup.2, together with the atoms to which they are bound, form an optionally substituted C.sub.6-14aryl group; A.sup.3, if present, is selected from H and optionally substituted C.sub.1-4alkyl; R.sup.N is selected from H and optionally substituted C.sub.1-4alkyl; one of B.sup.1, B.sup.2, B.sup.3, B.sup.4, and B.sup.5 is a group of formula XR.sup.X and the others are independently selected from H and R.sup.B; wherein each R.sup.B is independently selected from halogen, CF.sub.3, R, OH, OR, OCF.sub.3, C(O)OH, C(O)OR, C(O)R, OC(O)R, NH.sub.2, NHR, NR.sub.2, NO.sub.2, C(O)NH.sub.2, C(O)NHR, C(O)NR.sub.2, S(O)R, S(O).sub.2R, S(O).sub.2NR.sub.2, or CN; X is selected from a covalent bond or C.sub.1-3alkylene; R.sup.X is selected from H, R.sup.XX or R.sup.XY; wherein: R.sup.XX is halogen, CF.sub.3, OH, OR, OCF.sub.3, C(O)OH, NO.sub.2, NH.sub.2, NHR, NR.sub.2, C(O)NH.sub.2, C(O)NR.sub.2, S(O)R, S(O).sub.2R, S(O).sub.2NR.sub.2, or CN; and R.sup.XY is a group of formula -L.sup.X-R.sup.YY; wherein L.sup.X is selected from: NHC(O)O, NHC(O)NH, NHC(O), NRC(O), OC(O)NH, OC(O)O, O(C=O), C(O)NH, C(O)O, and C(O); and R.sup.YY is selected from C.sub.1-4alkyl, C.sub.3-6cycloalkyl, C.sub.6-14aryl, -L.sup.Y-C.sub.6-14aryl, -L.sup.Y-OC.sub.1-4aryl, C.sub.5-6heteroaryl, -L.sup.Y-C.sub.5-6heteroaryl, -L.sup.Y-OC.sub.5-6heteroaryl, -L.sup.Y-O-L.sup.Y-C=N, -L.sup.Y-O-L.sup.Y-C=R, -L.sup.Y-O-L.sup.Y-CCH, -L.sup.Y-O-L.sup.Y-NR.sup.NC(O)R, -L.sup.Y-O-L.sup.Y-NHC(O)R, -L.sup.Y-O-L.sup.Y-NRC(O)R, -L.sup.Y-CN, and -L.sup.Y-C=R, -L.sup.Y-CCH, wherein -L.sup.Y- is C.sub.1-3alkylene and wherein each of said groups is optionally substituted; R.sup.L is selected from halogen, R.sup.LL, CF.sub.3, OH, OR.sup.LL, NO.sub.2, NH.sub.2, NHR.sup.LL, NR.sub.2, NHC(O)R.sup.LL, NHC(O)OR.sup.LL wherein R.sup.LL is selected from C.sub.1-4alkyl, C.sub.3-6cycloalkyl, -Ph, -L.sup.L-Ph, C.sub.5-6heteroaryl, -L.sup.L-C.sub.5-6heteroaryl wherein -L.sup.L- is C.sub.1-3alkylene. and wherein each R is independently C.sub.1-4alkyl.

2. The compound described in claim 1, for use in a method of treatment of a disease or condition in an animal that can be treated by preventing or reducing microbial translocation through the epithelial barrier of the animal.

3. The compound for use according to claim 1 or claim 2, wherein the epithelial barrier is the gastrointestinal epithelial barrier.

4. The compound for use according to claim 3, wherein the intestinal epithelial barrier is the intestinal epithelial barrier.

5. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of a microbial infection in an animal.

6. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of febrile neutropenia in an animal.

7. The compound for use of any one of the above claims, wherein the animal has been or is being treated for cancer.

8. The compound for use of any one of the above claims, wherein the animal is the recipient of an organ transplant, optionally wherein the animal is immunocompromised.

9. The compound for use of any one of the above claims, wherein the animal has a low neutrophil count.

10. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of sepsis in an animal.

11. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of bacteremia or Fungemia in an animal.

12. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of mucositis in a subject.

13. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of microbiome dysbiosis in an animal.

14. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of a condition selected from: Atopic Dermatitis, Asthma, Allergic Rhinitis, Chronic rhinosinusitis, Eosinophilic Esophagitis, Meningitis, COPD, Periodontitis Bronchitis, Eczema, Inflammatory Bowel Disease, Coeliac Disease, Leaky Gut Syndrome, Alzheimer Disease, Parkinson Disease, Chronic Depression, Autism, Diabetes, Obesity, Non-Alcoholic Steatohepatitis, Autoimmune Hepatitis, Liver Cirrhosis, Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosus, Ankylosing Spondylitis, Intestinal Tissue Inflammation, Non alcoholic fatty liver disease and Necrotizing Enterocolitis.

15. The compound described in any one of the above claims, for use in a method of treatment and/or prevention of diarrhoea, for example traveller's diarrhoea.

16. The compound for use according to any one of the above claims, wherein administration of the compound improves or restores epithelial barrier function in the animal, and/or prevents or reduces microbial translocation through the epithelial barrier of the animal.

17. The compound for use according to claim 16, wherein the compound is capable of preventing microbial translocation from the gut of the animal to the kidney, liver, spleen or other organs and vascular beds via the circulatory system.

18. The compound for use according to claim 16, wherein the compound is capable of improving, restoring or maintaining tight junction function of the epithelial barrier of the animal.

19. The compound for use in a method of treatment and/or prevention of a microbial infection according to any one of the above claims, wherein the microbial infection is selected from the group consisting of bacterial, viral, prion, protozoal and fungal infections.

20. The compound for use according to claim 19, wherein said microbial infection is caused by a microbial species of a genus selected from the list consisting of: Yersenia, Salmonella, Shigella, Campylobacter, Clostridium; Heliobacter; Mycobacterium, Pseudomonas, Haemophilus, Moraxella, Escherichia, Neisseria, Streptococcus, Staphyllococcus, and Norovirus.

21. The compound for use according to claim 19, wherein said microbial infection is caused by Klebsiella pneumonia, Escherichia coli, Enterobacter spp., Serratia spp., Proteus spp., Providencia spp., Morganella spp., Enterococcus faecium, Staphylococcus aureus, Helicobacter pylori, Acinetobacter baumannii, Pseudomonas aeruginosa, Campylobacter (e.g. Campylobacter jejuni), Salmonella spp., Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, Shigella spp.

22. The compound for use according to claim 19, wherein said microbial infection is caused by Nairovirus, Marburg Virus, Ebola virus, Coronaviridae, Mammarenavirus, Henipavirus, Phlebovirus, Chikungunya, Alphavirus (Togavirus), Zika, and Dengue or other Flavivirus.

23. The compound for use according to claim 19, wherein said microbial infection is caused by Yersenia enterocolitica, E. coli, Clostridium difficile, Helicobacter pylori, Mycobacterium tuberculosis, Haemophilus influenza, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphyllococcus aureus, Group A and B Streptococcus, HIV, RSV, influenza virus, Herpes or Hepatitis viruses.

24. The compound for use according to claim 19, wherein said microbial infection is caused by a bacterial strain resistant to direct-acting antibiotic treatment.

25. The compound for use according to any one of the above claims, wherein the compound is a compound according to formula (Ia): ##STR00036##

26. The compound for use according to any one of the above claims, wherein A.sup.1 and A.sup.2, together with the atoms to which they are bound, form an optionally substituted phenyl, naphthalene or heteroaryl group.

27. The compound for use according to any one of the above claims, wherein A.sup.1 and A.sup.2, together with the atoms to which they are bound, form a phenyl group.

28. The compound for use according to any one of the above claims, wherein R.sup.N is H.

29. The compound for use according to any one of the above claims, wherein n is 0.

30. The compound for use according to any one of the above claims, wherein Q is Q1.

31. The compound for use according to any one of the above claims, wherein B.sup.3 is XR.sup.X.

32. The compound for use according to claim 30 or claim 31, wherein B.sup.1, B.sup.2, B.sup.4 and B.sup.5 are all H.

33. The compound for use according to any one of the above claims, wherein L is selected from (CH.sub.2).sub.m, C(O), NHC(O), and NRC(O).

34. The compound for use according to claim 32, wherein L is C(O).

35. The compound for use according to any one of the above claims, wherein X is a covalent bond.

36. The compound for use according to any one of claims 1-34, wherein X is C.sub.1-3alkylene.

37. The compound for use according to claim 36, wherein X is CH.sub.2.

38. The compound for use according to any one of the above claims, wherein R.sup.X is R.sup.XY.

39. The compound for use according to any one of the above claims, wherein Lx is independently: NHC(O)O, NHC(O), NRC(O), or O(C=O).

40. The compound for use according to claim 38, wherein L.sup.X is independently NHC(O) or NHC(O)O.

41. The compound for use according to any one of the above claims, wherein R.sup.YY is independently: -L.sup.Y-O-L.sup.Y-CH, -L.sup.Y-O-L.sup.Y-NHC(O)R or -L.sup.Y-CCH.

42. The compound for use according to any one of claims 1-40, wherein R.sup.YY is -L.sup.Y-C.sub.5-6heteroaryl.

43. The compound for use according to any one of the above claims, wherein -L.sup.Y- is CH.sub.2.

44. The compound for use according to any one of claims 1 to 40, wherein R.sup.YY is substituted with one or more substituents selected from: F, Cl, Br, I, R, CF.sub.3, OH, OR, OCF.sub.3, NO.sub.2, -L.sup.YY-OH, -L.sup.YY-OR, NH.sub.2, NHR, NR.sub.2, -L.sup.YY-NH.sub.2, -L.sup.YY-NHR, -L.sup.YY-NR.sub.2, CO.sub.2H, CO.sub.2R, -L.sup.YY-CO.sub.2H, -L.sup.YY-CO.sub.2R, -Ph, and -L.sup.YY-Ph-, wherein L.sup.YY is C.sub.1-3alkylene.

45. The compound for use according to any one of the above claims, wherein the compound is selected from: ##STR00037## and Pyridin-3-ylmethyl (4-((2-aminophenyl)-carbamoyl)benzyl)carbamate (Entinostat): ##STR00038##

46. The compound for use according to any one of the above claims, wherein the method comprises administration of the compound to the animal in an effective amount of the compound, wherein the weekly or daily dosage is between 10 g to about 1 g which is optionally split into doses given 1, 2 or 3 times.

47. The compound for use according to claim 46 wherein the dosage is between 0.025 mg and 500 mg.

48. The compound for use according to claim 47, wherein the dosage is between 0.1 mg and 250 mg.

49. The compound for use according to any one of claims 46-48, wherein the compound is administered at a dose of at least 5 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, or at least 100 mg/kg.

50. The compound for use according to claim 49, wherein the compound is administered at a dose of at least 50 mg/kg.

51. The compound for use according to any one of claims 46-50, wherein the compound is administered at a dose of less than 150 mg/kg.

52. The compound for use according to any one of the above claims, wherein the treatment is a combination treatment, wherein the compound is used in combination with any one or more of: an antibiotic; isoleucine or active isomers or analogs thereof; a vitamin D type compound.

53. The compound for use according to any one of the above claims, wherein the compound is present in a pharmaceutical composition comprising as an active ingredient said compound, in addition to at least one pharmaceutically acceptable excipient.

54. The compound for use according to claim 53, wherein the pharmaceutical composition is formulated as an oral dosage form.

55. The compound for use according to claim 54, wherein the oral dosage form is selected from a tablet, a capsule, a solution, a suspension, a powder, a paste, an elixir, a syrup, and a lozenge.

56. The compound for use according to claim 54 or claim 55, wherein the oral dosage form comprises in the range of about 0.01-1000 mg of said active ingredient.

57. The compound for use according to claim 53, wherein the pharmaceutical composition is formulated as an inhalation dosage form.

58. The compound for use according to any one of the above claims, wherein the method comprises: (1) administration to the animal of an antibiotic for 1 or 2 days with or without the compound; followed by (2) administration to the animal of an effective amount of the compound for a further 2, 3, 4, 5 or more days.

59. A method of treatment as described in any one of the above claims.

60. Use of a compound described in any one of the above claims in the preparation of a medicament for use in a method of treatment as described in any one of the above claims.

61. A compound, use or method as claimed in any one of the preceding claims wherein the animal is a mammal, preferably wherein the animal is a human.

62. A compound, use or method as claimed in any one of the preceding claims wherein the animal is selected from fish, dogs, cats, cows, horses, deer and poultry including hen, turkey, ducks, geese, and household pets such as birds and rodents.

63. A compound selected from: ##STR00039##

Description

FIGURES

[0331] FIG. 1: E. coli mouse infection model. CFU measured in tissue: colon, ileum, liver, spleen. Statistical analysis was performed by comparing all conditions to the vehicle-treated group and using one-way ANOVA with Dunnett post-hoc test, *** p<0.001, **** p<0.0001, ns non-significant.

[0332] FIG. 2: Induction test. Compounds 2.1, 2.2 and 2.3 induced expression of ProLL-37-luciferase fusion protein in HT-29 CampLuc MN8 reporter cell line after 24h. Corresponding concentrations of compound 1 (2-128 M) and Entinostat (2.5 M) were used as positive controls. The results are presented as an average of luminescence signal relative to control (untreated cells; value 1) from three independent experimentsSD. Statistical analysis was performed by comparison of each compound-treatment conditions to the corresponding vehicle control using two-way ANOVA with Dunnett post-hoc test. Only significant changes were indicated, ** p<0.01, *** p<0.001, **** p<0.0001. Entinostat treatment (Entino) was compared to control by using t-test, ##p<0.01.

[0333] FIG. 3: Schematic protocol for neutropenia mouse model

[0334] FIG. 4: CFU measured in blood, liver, kidney and spleen in a mouse model of febrile neutropenia, after administration with the listed compounds at the listed concentrations.

[0335] FIG. 5: Body weights measured in a mouse model of febrile neutropenia (with reference to Nave control), after administration with the listed compounds at the listed concentrations.

[0336] FIG. 6: Amplification plot for samples from RT-PCT example.

[0337] FIG. 7: .sup.1H NMR spectrum of compound 1.

[0338] FIG. 8: LC/MS of compound 1.

[0339] FIG. 9: .sup.1H NMR spectrum of compound 2.1.

[0340] FIG. 10: LC/MS of compound 2.1.

[0341] FIG. 11: .sup.1H NMR spectrum of compound 2.2.

[0342] FIG. 12: LC/MS of compound 2.2.

[0343] FIG. 13: .sup.1H NMR spectrum of compound 2.3.

[0344] FIG. 14: LC/MS of compound 2.3.

EXAMPLES

Methods and Materials

[0345] MN8CampLuc cells were handled according to Nyln et. al. with the following exception when predifferention of cells were performed before induction:

[0346] Cell seeding was performed in medium where glucose was exchanged for galactose (5 mg/ml), which is known to promote differentiation in colon epithelial cells (Pinto, M., M. D. Appay, P. Simon-Assman, G. Chevalier, N. Dracopoli, J. Fogh, and A. Zweibaum, 1982, Biol. Cell., 44:193-196) Cells were then allowed to grow for 72 hours before stimulation with test compounds.

[0347] RT-PCR experiments were performed according to Nylen et. al. (Nylen F, Miraglia E, Cederlund A, Ottosson H, Stromberg R, Gudmundsson G H, Agerberth B. 2013. Boosting innate immunity: Development and validation of a cell-based screening assay to identify LL-37 inducers. Innate Immun.).

[0348] RT-PCR experiments for expression of marker genes for autophagy in HEK-293 cells were measured by real-time PCR. Data were normalized by the expression of the 18s rRNA housekeeping gene. For the immunofluorescence spectroscopy experiments HEK-293 cells were fixed after treatment with the inducers or control. The cells were then stained with DAPI to visualize the nuclei (blue), and immunolabeled with the anti-LC3, followed by the addition of Alexa-fluor 488 (green). Scale bar=10 m.

[0349] All reagents and solvents (analytical grade) were purchased from commercial resources and were used without further purification. The NMR spectra were collected on a Bruker DRX-400 spectrometer (400 MHz for .sup.1H and 101 MHz for .sup.13C) with the residual solvent signal as chemical shift reference. Mass spectra were recorded on a Micromass LCT (ESI-TOF) mass spectrometer. Pyridin-3-ylmethyl (4-((2-aminophenyl)carbamoyl)benzyl)carbamate (5, Entinostat) and N1-hydroxy-N8-phenyloctanediamide (12, Vorinostat) was purchased from LC laboratories (Woburn, MA, USA), N-(4-Methoxybenzyl)-1,2-benzenediamine (16) from Fluorochem Ltd (Hadfield, UK) and Trichostatin A (19) from Sigma-Aldrich Sweden AB (Stockholm, Sweden).

Example 1. Synthesis of Compounds Relating to the Invention

[0350] Exemplary syntheses are provided below. Compounds of the invention may also be synthesised by other methods known in the art.

[0351] Compound 1 may be synthesised as follows:

##STR00029##

[0352] Step A: To a solution of 2 (1 equiv.) in acetonitrile was added CDI (1.2 equiv.). The reaction mixture was stirred for 40 min at r.t. and 1 (1 equiv.) was added. The resulting mixture was stirred overnight at 40 C., evaporated under reduced pressure, and diluted with ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium sulfate and concentrated in vacuum. Purification of the residue via column chromatography on silica gel afforded 3.

[0353] Step B: To a solution of 3 in dichloromethane was added TFA (10 equiv.), and the reaction mixture was stirred overnight r.t. and evaporated under reduced pressure. The residue was crystallized from hexane to obtain 4.

[0354] Step C: To a solution of 4 in DMF was added triethylamine (2 equiv.) and HATU (1.1 equiv.), followed by 5 (1 equiv.). The reaction mixture was stirred overnight at r.t., diluted with water, and extracted ethyl acetate. The combined organic layers were washed with water, dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduced pressure to afford 6 which was used in the next step without further purification.

[0355] Step D: To a solution of 6 in dichloromethane was added TFA (10 equiv.), and the reaction mixture was stirred overnight r.t. and evaporated under reduced pressure. The residue was diluted with water and pH was adjusted to 8 with aq. solution of sodium bicarbonate. The precipitated product was isolated by simple filtration and washed with water and dried to obtain compound 1.

[0356] Analytical data for compound 1 is shown in FIGS. 7 and 8.

[0357] Compound 2.1 may be synthesised as follows:

##STR00030##

[0358] Step F: To the solution of 6 (3 g, 26.3 mmol) in dry DMF (50 mL) were added HATU (10.9 g, 28.6 mmol) and DIPEA (11.1 g, 85.8 mmol) and the mixture was stirred at r.t. for 30 min. Then 5 (6.16 g, 28.6 mmol, HCl salt) was added and the reaction mixture was stirred overnight at r.t. The solvent was evaporated, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL), aq. citric acid (10%, 100 mL) and aq. NaHCO.sub.3 (100 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain crude 7. After column chromatography (H:EA=1:1) pure ester 7 was obtain (5.55 g, 76%).

[0359] Step G: To a solution of 7 (5.55 g, 20.2 mmol) in MeOH (100 mL) was added NaOH (1.2 g, 30 mmol) in water (5 mL) and the reaction mixture was stirred overnight at r.t. The solvent was evaporated, the residue was diluted with water, acidified by citric acid, and extracted with EtOAc (3100 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain pure acid 8 (4.65 g, 88%).

[0360] Step H: To a solution of 8 (4.65 g, 17.8 mmol) in dry THF (50 mL) were added HATU (7.45 g, 19.6 mmol) and DIPEA (7.6 g, 58.7 mmol) and the mixture was stirred at r.t. for 30 min. Then 9 (4.1 g, 19.6 mmol) was added and the reaction mixture was stirred overnight at r.t. The solvent was evaporated, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL), aq. citric acid (10%, 100 mL) and aq. NaHCO.sub.3 (100 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain crude 10. After column chromatography (H:EA=1:4) pure ester 10 was obtain (5.6 g, 70%).

[0361] Step I: To a solution of 10 (5.6 g, 12.4 mmol) in dry dioxane (60 mL) was added HCl\Dioxane (60 mL) and the reaction mixture was stirred overnight at r.t. Then the resulting mixture was diluted with MTBE (100 mL), the solid was filtered and dried under reduced pressure to obtain compound 2.1 as HCl salt.

[0362] Step J: Compound 2.1 HCl salt (2 g) was dissolved in aq. NaHCO.sub.3 (50 mL) and extracted with DCM (330 mL). Organic layers were dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain compound 2.1.

[0363] Analytical data for compound 2.1 is shown in FIGS. 9 and 10.

[0364] Compound 2.2 may be synthesised as follows:

##STR00031##

[0365] Step K: To a solution of 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid (3 g, 13.7 mmol) in dry DMF (50 mL) were added HATU (5.7 g, 15 mmol) and DIPEA (5.8 g, 45.2 mmol) and the mixture was stirred at r.t. for 30 min. Then 5 (3.23 g, 15 mmol, HCl salt) was added and the reaction mixture was stirred overnight at r.t. The solvent was evaporated, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL), aq. citric acid (10%, 100 mL) and aq. NaHCO.sub.3 (100 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain crude 11. After column chromatography (H:EA=1:1) pure ester 11 was obtain (3 g, 57.5%).

[0366] Step L: To a solution of 11 (3 g, 7.89 mmol) in dry dioxane (30 mL) was added HCl\Dioxane (30 mL) and the reaction mixture was stirred overnight at r.t. The resulting mixture was diluted with MTBE (100 mL), the solid was filtered and dried under reduced pressure to obtain 12 as HCl salt.

[0367] Step M: To a suspension of 12 (1.8 g, 5.7 mmol) in dry THF (30 mL) was added TEA (2.3 g, 22.8 mmol) and the mixture was cooled to 0 C. Then propionyl chloride (0.63 g, 6.8 mmol) was added dropwise and the reaction mixture was stirred overnight at r.t. The solvent was evaporated, the residue was dissolved in DCM (50 mL) and washed with citric acid. The organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain pure 13 (1.9 g, 90%).

[0368] Step N: To a solution of 13 (1.9 g, 5.7 mmol) in MeOH (30 mL) was added NaOH (0.34 g, 8.5 mmol) in water (1 mL) and the reaction mixture was stirred overnight at r.t. The solvent was evaporated, the residue was diluted with water, acidified with citric acid, and extracted with EtOAc (3100 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain pure acid 14 (0.4 g, 25%).

[0369] Step O: To a solution of 14 (0.4 g, 1.2 mmol) in dry THF (10 mL) were added HATU (0.5 g, 1.3 mmol) and DIPEA (0.52 g, 4 mmol) and the mixture was stirred at r.t. for 30 min. Then 9 (see 9 in synthesis for compound 2.1) (0.27 g, 1.3 mmol) was added and the reaction mixture was stirred overnight at r.t. The solvent was evaporated, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL), aq. citric acid (10%, 100 mL) and aq. NaHCO.sub.3 (100 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain pure 15 was obtain (0.5 g, 75%).

[0370] Step P: To a solution of 15 (0.5 g, 0.98 mmol) in dry dioxane (10 mL) was added HCl\Dioxane (10 mL) and the reaction mixture was stirred overnight at r.t. Then the resulting mixture was diluted with MTBE (100 mL), the solid was filtered and dried under reduced pressure to obtain compound 2.2 as HCl salt.

[0371] Analytical data for compound 2.2 is shown in FIGS. 11 and 12.

[0372] Compound 2.3 may be synthesised as follows:

##STR00032##

[0373] Step A: To a solution of 2 (3 g, 21.7 mmol) in dry THF (50 mL) were added HATU (9.1 g, 23.9 mmol) and DIPEA (9.3 g, 71.6 mmol) and the mixture was stirred at r.t. for 30 min. Then tert-butyl (2-aminophenyl)carbamate (1) (5 g, 23.9 mmol) was added and the reaction mixture was stirred overnight at r.t. After that, the solvent was evaporated, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL), ag. citric acid (10%, 100 mL) and aq. NaHCO.sub.3 (100 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced (2.65 g) pressure to obtain crude 3. After column chromatography (H:EA=1:1) pure 3 was obtain (2.65 g).

[0374] Step B: To a solution of 3 (2.65 g, 8 mmol), 4 (1.32 g, 12 mmol) and DMAP (0.72 g, 5.9 mmol) in dry DMF (30 mL) was added DCC (2.43 g, 12 mmol) and the reaction mixture was stirred overnight at room temperature. Then the resulting mixture was quenched with water and extracted with EtOAc (350 mL). Organic layers were combined, washed with aq. NaHCO.sub.3 and brine. Then dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure. The crude was purified by column chromatography (CHCl.sub.3:MeOH=24:1) to obtain pure 5 (1.5 g).

[0375] Step C: To a solution of 5 (1.5 g, 3.55 mmol) in dry dioxane (10 mL) was added HCl\Dioxane (20 mL) and the reaction mixture was stirred overnight at r.t. Then the resulting mixture was diluted with MTBE (50 mL), the solid was filtered and dried under reduced pressure to obtain compound 2.3 as HCl salt. The solid was dissolved in aq. NaHCO.sub.3 and extracted with DCM (330 mL). Organic layers were dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to obtain compound 2.3.

[0376] Analytical data for compound 2.3 is shown in FIGS. 13 and 14.

Example 2. E. coli Mouse Infection Model

[0377] Compound 1 was administered orally to a mouse model of E. coli infection at 25 mg/kg, 73.5 mk/kg and 50 mg/kg. Compound 1 achieved the study goal of lowering Colony Unit Forming count in colon tissue by a factor of 3 log.sub.10 compared to vehicle treated animals.

[0378] Unexpectedly, it was found that 1) Colony Forming Unit counts in liver and spleen turned out to be higher than expected (on the order of 2-3 log.sub.10), and 2) Compound 1 had the unexpected effect of completely knocking out translocation of bacteria to these organs.

Example 3: Evaluation of Further Compounds of the Invention

[0379] Further compounds were newly designed (compounds 2.1, 2.2 and 2.3), and were synthesised variants of compound 1.

[0380] Compounds 2.1, 2.2 and 2.3 were tested for induction of antimicrobial peptides in a human cell line. The experiment yielded the dose dependent fold induction for expression of CAMP gene (encoding human cathelicidin), as shown in FIG. 2.

Example 4: Comparison of Compound 1 to HO13 and HO56

[0381] Compound 1 was selected as a particularly preferred embodiment of the invention, after comparison with HO53 and HO56 (Myszor, I. T., Parveen, Z., Ottosson, H. et al. Novel aroylated phenylenediamine compounds enhance antimicrobial defense and maintain airway epithelial barrier integrity. Sci Rep 9, 7114 (2019). https://doi.org/10.1038/s41598-019-43350-z):

##STR00033##

[0382] Table 1 below depicts comparison data for the three compounds. An important characteristic of compound 1 vs HO53 and HO56 is that it unexpectedly displays high affinity (permeability) in a Caco-2 cell study. Caco-2 cells are used as a model of the intestinal epithelial barrier. The results thus show that the compounds of the invention are particularly beneficial in treating diseases that can be treated by improving or restoring gastrointestinal barrier function, and/or preventing or reducing microbial translocation through the gastrointestinal barrier.

TABLE-US-00002 TABLE 1 Compound HO53 HO56 Compound 1 Caco-2 Permeability Low Low High A-B dir. Active efflux Yes Yes No Plasma stability Mouse Low High Low Plasma stability Rat High High High Stability SGD, High High High SIF w. Enzymes Dose Esc. Tox >100 mg/kg 10 mg/kg 100 mg/kg mouse iv Dose Esc. Tox >100 mg/kg >200 mg/kg 50 mg/kg mouse PO 7 Days PO once daily >50 mg/kg >50 mg/kg >50 mg/kg MTD - mouse 7 Days PO twice daily 50 mg/kg MTD - mouse Bioavailability rat 3% 16% 1% C_max IV - rat 21 M 26 M 177 M C_max PO - rat 0.09 M 1.2 M 3.3 M V-d IV - rat 1700 ml/kg 3070 ml/kg 565 ml/kg Elimination T (min) 35 132 34 PO - rat Elimination T (min) 126 94 66 PIV - rat PO single 5 mg/kg 0.9 M mouse - Ileum C-max PO single 15 mg/kg 2.0 M mouse - Ileum C-max PO single 50 mg/kg 7.1 M mouse - Ileum C-max

Example 5: Mouse Model Results

[0383] A mouse model was prepared to mimic chemically induced febrile neutropenia. A schematic of the experimental model is depicted in FIG. 3.

[0384] The results of this experiment are shown in Tables 2-7 and FIGS. 4 and 5. Certain observations include: [0385] Animals were apparently normal in Nave and compound 1 and Entinostat treated groups. Animals in vehicle control observed lethargic on day 7 [0386] Clinical signs were consistent with efficacy. [0387] CFU Load in blood was BLOQ in all the groups on day 5 & 6 [0388] compound 1 showed significant dose dependent antibacterial effect in liver and blood when compared to their corresponding vehicle controls (p<0.05) on day 7 [0389] Bacteria was BLOQ in animals treated with Entinostat and compound 1 (50 mg/kg) on day 7 [0390] Colonies recovered from plates were confirmed to Enterotoxigenic Escherichia coli by RT-PCR.

TABLE-US-00003 TABLE 2 Efficacy of compound 1 against Intestinal Bacterial Translocation of Enterotoxigenic Escherichia coli (ETEC) (ATCC 35401, H10407) in a Neutropenic Mouse Model. Body weights Log.sub.10 CFU/g.sup.#[Mean SD] (Final Day) Group Blood Liver Spleen Kidneys (Mean SD) Nave [none] BLOQ BLOQ BLOQ BLOQ 28.2 0.92* Vehicle Control DMSO: 2.15 0.31 3.35 0.26 3.07 0.3.sup. 3.04 0.39.sup. 23.3 0.95 PEG400: dH2O (20%:50%:30%, v/v) PO, twice daily(q12h), 7 days Entinostat (Positive BLOQ BLOQ BLOQ BLOQ 26.5 1.58* control) 1 mg/kg, PO, twice daily(q12h), 9 days Compound 1 1.9 0* 2.58 0.32* 3.05 0.3.sup.ns 2.83 0.29.sup.ns 25.2 1.69* 5 mg/kg, PO, twice daily(q12h), 9 days Compound 1 BLOQ BLOQ BLOQ BLOQ 26.1 1.20* 50 mg/kg, PO, twice daily(q12h), 9 days

TABLE-US-00004 TABLE 3 Clinical observations in a mouse model of febrile neutropenia (with reference to Nave control), after administration with the listed compounds at the listed concentrations. Clinical observation (Mean Score) Entinostat Compound 1, Compound 1, Vehicle, [1 mg/kg, 5 mg/kg, 50 mg/kg, PO, twice PO, twice PO, twice PO, twice Clinical Nave daily(q12h), daily(q12h), daily(q12h), daily(q12h), Days signs [none] 9 days 9 days] 9 days 9 days Day (2) Rough coat 0.0 0.0 0.0 0.0 70.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.0 0.0 0.0 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (1) Rough coat 0.0 0.0 0.0 0.0 0.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.0 0.0 0.0 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (0) Rough coat 0.0 0.0 0.0 0.0 0.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.0 0.0 0.0 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (1) Rough coat 0.0 0.0 0.0 0.0 0.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.0 0.0 0.0 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (2) Rough coat 0.0 0.0 0.0 0.0 0.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.0 0.0 0.0 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (3) Rough coat 0.0 0.0 0.0 0.0 0.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.0 0.0 0.0 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (4) Rough coat 0.0 0.0 0.0 0.0 0.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.0 0.0 0.0 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (5) Rough coat 0.0 0.6 0.1 0.5 0.0 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.3 0.0 0.1 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (6) Rough coat 0.0 1.0 0.1 0.7 0.4 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.5 0.0 0.4 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 Day (7) Rough coat 0.0 1.0 0.2 1.0 0.4 Hunched 0.0 0.0 0.0 0.0 0.0 Posture Lethergy 0.0 0.7 0.0 0.5 0.0 hyperpnea 0.0 0.0 0.0 0.0 0.0 0 = No or absent; 1 = Yes or Present

TABLE-US-00005 TABLE 4 Liver-Raw data on Vancomycin (32 g/ml) plates, Log10 CFU/g Vehicle Control DMSO: Entinostat Compound 1, Compound 1, PEG400: dH.sub.2O 1 mg/kg, 5 mg/kg, 50 mg/kg, PO, twice PO, twice PO, twice daily(q12h), Nave daily(q12h), daily(q12h), daily(q12h), PO, twice (none) 9 days 9 days 9 days 9 days BLOQ 3.49 BLOQ 2.41 BLOQ BLOQ 4.00 BLOQ 2.67 BLOQ BLOQ 3.41 BLOQ 2.41 BLOQ BLOQ 3.33 BLOQ 3.42 BLOQ BLOQ 3.25 BLOQ 2.33 BLOQ BLOQ 3.01 BLOQ 2.39 BLOQ BLOQ 3.27 BLOQ 2.39 BLOQ BLOQ 3.24 BLOQ 2.53 BLOQ BLOQ 3.29 BLOQ 2.61 BLOQ BLOQ 3.22 BLOQ 2.57 BLOQ

TABLE-US-00006 TABLE 5 Spleen -Raw data on Vancomycin (32 g/ml) plates, Log10 CFU/g Vehicle Control DMSO: Entinostat Compound 1, Compound 1, PEG400: dH.sub.2O 1 mg/kg, 5 mg/kg, 50 mg/kg, PO, twice PO, twice PO, twice PO, twice Nave daily(q12h), daily(q12h), daily(q12h), daily(q12h), (none) 9 days 9 days 9 days 9 days BLOQ 2.75 BLOQ 3.17 BLOQ BLOQ 2.89 BLOQ 3.04 BLOQ BLOQ 3.11 BLOQ 3.00 BLOQ BLOQ 2.78 BLOQ 3.51 BLOQ BLOQ 3.56 BLOQ 2.62 BLOQ BLOQ 3.02 BLOQ BLOQ BLOQ BLOQ 3.52 BLOQ 2.91 BLOQ BLOQ 2.75 BLOQ BLOQ BLOQ BLOQ 3.08 BLOQ BLOQ BLOQ BLOQ 3.23 BLOQ BLOQ BLOQ

TABLE-US-00007 TABLE 6 Kidney -Raw data on Vancomycin (32 g/ml) plates, Log10 CFU/g Vehicle Control DMSO: Entinostat Compound 1, Compound 1, PEG400: dH.sub.2O 1 mg/kg, 5 mg/kg, 50 mg/kg, PO, twice PO, twice PO, twice PO, twice Nave daily(q12h), daily(q12h), daily(q12h), daily(q12h), (none) 9 days 9 days 9 days 9 days BLOQ 3.40 BLOQ BLOQ BLOQ BLOQ 3.15 BLOQ 2.73 BLOQ BLOQ 3.89 BLOQ 3.23 BLOQ BLOQ 2.91 BLOQ 2.66 BLOQ BLOQ 2.59 BLOQ 2.94 BLOQ BLOQ 2.92 BLOQ 3.34 BLOQ BLOQ 3.06 BLOQ 2.68 BLOQ BLOQ 2.73 BLOQ 2.59 BLOQ BLOQ 3.04 BLOQ 2.54 BLOQ BLOQ 2.65 BLOQ 2.75 BLOQ

TABLE-US-00008 TABLE 7 Blood -Raw data on Vancomycin (32 g/ml) plates, Log10 CFU/g - on day 7 PI Vehicle Control DMSO: Entinostat Compound 1, Compound 1, PEG400: dH.sub.2O 1 mg/kg, 5 mg/kg, 50 mg/kg, PO, twice PO, twice PO, twice PO, twice Nave daily(q12h), daily(q12h), daily(q12h), daily(q12h), (none) 9 days 9 days 9 days 9 days BLOQ 2.30 BLOQ 1.90 BLOQ BLOQ 2.51 BLOQ 1.90 BLOQ BLOQ 1.90 BLOQ BLOQ BLOQ BLOQ 2.72 BLOQ BLOQ BLOQ BLOQ 1.90 BLOQ 1.90 BLOQ BLOQ 1.90 BLOQ 1.90 BLOQ BLOQ 1.90 BLOQ BLOQ BLOQ BLOQ 2.38 BLOQ BLOQ BLOQ BLOQ 2.08 BLOQ 1.90 BLOQ BLOQ 1.90 BLOQ BLOQ BLOQ

Example 6: RT-PCR Data

[0391] RNA Extraction: RNA from representative bacterial colonies on plates were extracted by Nucleo-Pore RNASure Mini Kit. Final volume of RNA eluted was 30 l. Further these sample RNAs were processed for qRT-PCR.

[0392] Quantitative real-time polymerase chain reaction (qRT-PCR): Equal volume of RNA obtained was used in setting up the qRT-PCR reaction and RNA-Direct SYBR Green Realtime PCR Master Mix kit was used. Further samples were analysed on a QuantStudio 3 Real-Time PCR System.

[0393] Results: As shown in Table 8 and FIG. 6, RT-PCR data, confirms colonies recovered from plates are Enterotoxigenic Escherichia coli.

TABLE-US-00009 TABLE 8 CFU/ml- Samples Ct Values #13 spleen 29.17 #34 spleen 30.24 #13 kidney 28.48 #14 kidney 29.69 #33 kidney 28.74 #36 kidney 29.69 #12 Liver 30.03 #34 Liver 29.69 # 14 Blood 26.58

Example 7: Summary of Experiments

[0394] Infecting mice through oral administration of a vancomycin resistant pathogenic strain of E. coli simplified the process of determining the mechanism(s) by which compound 1 exerts its effects on the pathogenic process. All bacteria grown and detected in these experiments are not commensal bacteria because they are killed and do not grow on bacteria plates that have vancomycin. [0395] Compound 1 is not acting as an antibiotic nor is it stimulating the production of innate antimicrobial agents to such an extent that they function as general antibiotics. This is evidenced by the fact that there still are viable vancomycin resistant E. coli present in the colon and ileum at the conclusion of the experiment (FIG. 1). The E. coli burden in the intestines is reduced by close to 3-logs compared to control, which indicates that the growth of the E. coli is attenuated, most likely through stimulation of innate antimicrobial peptide production. This is supported by in vitro data which demonstrates that compound 1 is not toxic to bacteria in culture and that innate antimicrobial peptides function like general antibiotics only at super physiological concentrations. [0396] FIG. 1 also demonstrates that the inhibition of vancomycin resistant E. coli translocation to the liver and spleen is not compound 1 dose dependent. In contrast, compound 1 shows dose dependence in the attenuation of E. coli growth in the colon and ileum. It is therefore unlikely that compound 1 is inhibiting E. coli translocation to these organs solely due to its ability to stimulate production of antibacterial peptides. The most likely explanation is the ability of compound 1 and related compounds to stimulate and strengthen the cell-cell contacts, as has been demonstrated by in vitro cell culture experiments. This is supported by the data presented in FIG. 4. That experiment was performed in the presence of cyclophosphamide, which is known to cause febrile neutropenia due to translocation of bacteria to vital organs. In FIG. 4, the lower concentration of compound 1 (5 mg/kg) is having little to no effect on bacterial translocation while the higher concentration (50 mg/kg) inhibits it completely. [0397] Taken together these data suggest two cooperating but separate mechanisms are responsible for the actions of compound 1 on E. coli pathogenicity in the intestines. First is the ability of the compound 1 to attenuate the growth of bacteria and secondly is its ability to inhibit the bacterial translocation.

Example 8: HDAC Inhibition Data

[0398] The purpose of the study is to determine the effects of Compound 1 on the enzymatic activities of recombinant human HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, and HDAC9 using in-vitro enzymatic assays.

[0399] Materials were as follows: [0400] HDAC Assay Buffer (BPS catalog number 50031) [0401] HDAC Assay Developer (BPS catalog number 50030) [0402] HDAC Substrate 3 (BPS number 50037) [0403] HDAC Class 2a Substrate 1 (BPS number 50040) [0404] TSA was purchased from Selleck (Houston, TX, Catalog number S1045) [0405] SAHA was purchased from Cayman Chemicals (Ann Arbor, MI, Catalog Number 10009929)

[0406] Compounds were as follows:

TABLE-US-00010 Compound Compound Stock Dissolving Test Range Intermediate I.D. Supplied Concentration Solvent (M) Dilution AKT-011 Solid 20 mM DMSO 1-200 10% DMSO in Assay Buffer SAHA* Solution 10 mM DMSO 0.003-100 10% DMSO in Assay Buffer TSA* Solution 10 mM DMSO 0.003-100 10% DMSO in Assay Buffer *Reference Compounds

[0407] Experimental conditions were as follows:

TABLE-US-00011 Enzyme Used Assay Catalog# Enzyme Lot# (ng/rxn) Substrate HDAC1 50051 200917-1 8 20 uM HDAC Substrate 3 HDAC2 50002 150701 12 20 uM HDAC Substrate 3 HDAC3 50003 190326 5 20 uM HDAC Substrate 3 HDAC4 50004 130828-G 1 2 uM HDAC Substrate 2A HDAC5 50005 201217-G3 30 2 uM HDAC Substrate 2A HDAC6 50006 220419-GC 20 20 uM HDAC Substrate 3 HDAC7 50007 150825A 2 2 uM HDAC Substrate 2A HDAC8 50008 161220 20 2 uM HDAC Substrate 2A HDAC9 50009 150520-G3 5 2 uM HDAC Substrate 2A

[0408] Assay conditions were as follows:

[0409] All of the compounds were dissolved in DMSO. A series of dilutions of the compounds were prepared with 10% DMSO in HDAC assay buffer and 5 l of the dilution was added to a 50 l reaction so that the final concentration of DMSO was 1% in all of reactions. The compounds were pre-incubated in duplicate at RT for 30 minutes in a mixture containing HDAC assay buffer, 5 g BSA, HDAC enzyme and a test compound. After 30 minutes, the enzymatic reactions were initiated by the addition of HDAC substrate to a final concentration of 20 M or 2 M. The enzymatic reaction proceeded for 30 minutes at 37 C. After enzymatic reactions, 50 l of 2HDAC Developer was added to each well for the HDAC enzymes and the plate was incubated at room temperature for an additional 15 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 microplate reader.

[0410] Data analysis was as follows:

[0411] HDAC activity assays were performed in duplicates at each concentration. The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(FFb)/(FtFb), where F=the fluorescent intensity in the presence of the compound. The values of % activity versus a series of compound concentrations were then plotted using non-linear regression analysis of Sigmoidal dose-response curve generated with the equation Y=B+(TB)/1+10{circumflex over ()}((Log EC50X)Hill Slope), where Y=percent activity, B=minimum percent activity, T=maximum percent activity, X=logarithm of compound and Hill Slope=slope factor or Hill coefficient. The IC50 value was determined by the concentration causing a half-maximal percent activity.

[0412] A summary of the effects of the compounds on HDAC activities is as follows:

TABLE-US-00012 IC.sub.50 (M) Enzymes AKT-011 SAHA TSA HDAC1 IC.sub.50 < 1 M 0.087 75% Inhib. @ 1 M HDAC2 ~ 1.2 0.39 HDAC3 IC.sub.50 1 M 0.087 61% Inhib. @ 1 M HDAC4 IC.sub.50 > 200 M 6.7 No Inhib. @ 200 M HDAC5 IC.sub.50 > 200 M 4.6 No Inhib. @ 200 M HDAC6 IC.sub.50 > 200 M 0.041 No Inhib. @ 200 M HDAC7 IC.sub.50 > 200 M 3.6 No Inhib. @ 200 M HDAC8 IC.sub.50 > 200 M 1.8 42% Inhib. @ 200 M HDAC9 IC.sub.50 > 200 M 4.0 No Inhib. @ 200 M

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