Amidine substituted analogues and uses thereof

Abstract

The present application relates to amidine substituted analogues as shown in formula I and uses thereof as an anti-inflammatory agent and/or for the treatment of an immunological disorder or of an inflammatory bowel disease including ulcerative colitis, Crohn's diseases, collagenous colitis and lymphocytic colitis. ##STR00001##

Claims

1. A compound of formula (I): ##STR00032## or a pharmaceutically acceptable salt thereof, wherein: W is a carbon atom or phenyl; V.sub.1, V.sub.2 and V.sub.3 are each independently C.sub.3-C.sub.12 alkyl, C.sub.3-C.sub.12 alkyl interrupted by —O—, —OC.sub.3-C.sub.12 alkyl or —OC.sub.3-C.sub.12 alkyl wherein the C.sub.3-C.sub.12 alkyl is interrupted by —O—; and A.sub.1, A.sub.2 and A.sub.3 are each independently —C(═NH)—NH.sub.2 or —C(═NH)—NHOH; provided that when V.sub.1(V.sub.2 and V.sub.3 are —(CH.sub.2).sub.1-6-; then at least one of A.sub.1, A.sub.2 and A.sub.3 is C(═NH)—NHOH.

2. The compound according to claim 1, wherein W is phenyl.

3. The compound according to claim 1, wherein W is a carbon atom.

4. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each independently C.sub.3-C.sub.12 alkyl or C.sub.3-C.sub.12 alkyl interrupted by —O—.

5. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each independently C.sub.5-C.sub.12 alkyl or C.sub.5-C.sub.12 alkyl interrupted by —O—.

6. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each C.sub.3-C.sub.12 alkyl.

7. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each C.sub.5-C.sub.12 alkyl.

8. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each independently —OC.sub.3-C.sub.12 alkyl or —OC.sub.3-C.sub.12 alkyl interrupted by —O—.

9. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each independently —OC.sub.5-C.sub.12 alkyl or —OC.sub.5-C.sub.12 alkyl interrupted by —O—.

10. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each —OC.sub.3-C.sub.12 alkyl.

11. The compound according to claim 1, wherein V.sub.1, V.sub.2 and V.sub.3 are each —OC.sub.5-C.sub.12 alkyl.

12. The compound according to claim 1, wherein the compound of formula (I) is represented by: ##STR00033## wherein W, V.sub.1, V.sub.2, V.sub.3, A.sub.1, A.sub.2 and A.sub.3 are as defined in claim 1.

13. The compound according to claim 1, where the compound is: ##STR00034## ##STR00035##

14. A pharmaceutical composition comprising a compound according to claim 1 with a pharmaceutically acceptable carrier, diluent and excipient.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents the ALT levels 6 hours post liver injury induction by GalN/LPS.

(2) FIG. 2 represents the survival at 8 and 23 hours post liver injury induction by GalN/LPS.

(3) FIG. 3 represents ALT Activity, 6 hours post liver injury (Study b).

(4) FIG. 4 ALT Activity, 6 hours post liver injury (Study c).

(5) FIG. 5 represents the accumulated disease index at day 7.

EXAMPLES

(6) As used herein, the following abbreviations may have the following meanings:

(7) TABLE-US-00002 Abbreviation Term AcOH Acetic acid Approx. Approximately Aq. Aqueous CHCl.sub.3 Chloroform Cs.sub.2CO.sub.3 Cesium carbonate d Day(s) DCM Dichloromethane DDQ 2,3-dichloro-5,6-dicyano-p-benzoquinone DIAD Diisopropylazodicarboxylate DMAP Dimethylaminopyridine DME 1,2-dimethoxyethane DMF N,N-dimethyl formamide EtOAc Ethyl acetate h Hour(s) HATU (dimethylamino)-N,N-dimethyl(3H- [1,2,3]triazolo[4,5-b]pyridin-3- yloxy)methaniminium hexafluorophosphate HCl hydrochloric acid KOtBu Potassium tert-butoxide LC-MS Liquid chromatography mass spectrum min Minute(s) MeCN Acetonitrile MeOH Methanol MgSO.sub.4 Magnesium sulfate MsCl Methanesulfonyl chloride N.sub.2 Nitrogen NaBH.sub.4 Sodium borohydride NaHCO.sub.3 Sodium bicarbonate NaOH Sodium hydroxide Na.sub.2SO.sub.4 Sodium sulfate NMR Nuclear magnetic resonance Pd(OAc).sub.2 Palladium acetate PPh.sub.3 Triphenylphosphine Prep Preparative pTSA p-Toluenesulfonic acid rt Room temperature SFC Supercritical fluid chromatography TEA Triethylamine THF Tetrahydrofuran TFA Trifluoroacetic acid TLC Thin layer chromatography
General:

(8) All temperatures are in degrees Celsius (° C.) and are uncorrected.

Example 1: Synthesis of Trihexamidine Formate (Compound #1)

(9) ##STR00010##

(10) Step 1:

(11) To 11.9 g (100 mM) of cyanophenol and potassium carbonate (20.7 g, 150 mM) was added CH.sub.3CN (200 mL) and 1,6-dibromohexane (76 mL, 500 mM) and the mixture was refluxed over weekend. The mixture was cooled to room temperature, the solid was filtered off, washed with EtOAc, the solvent evaporated and the residue purified by combiflash to afford 23.95 g of A.

(12) ##STR00011##

(13) Step 2:

(14) To the triphenol (1.05 g, 8.3 mM) was added DMF (30 mL), cesium carbonate (13.6 g, 41.6 mM) and the bromo A (11.7 g, 41.6 mM). The mixture was heated at 65° C. over night. After cooling to room temperature the mixture was diluted with water, extracted with EtOAc (2×), washed with water (3×), brine, dried with Na.sub.2SO.sub.4, filtered and the solvent evaporated, after purification on the combiflash compound B (4.1 g) was obtained.

(15) ##STR00012##

(16) Step 3:

(17) A mixture of tricyano B (271 mg, 0.37 mM), hydroxylamine hydrochloride (155 mg, 2.23 mM) and triethylamine (0.31 mL, 2.23 mM) in ethanol 15 mL was refluxed overnight. The solvent was evaporated, the residue purified on the combiflash to give 288 mg of C.

(18) ##STR00013##

(19) Step 4:

(20) C (288 mg, 0.34 mM) in AcOH (5 mL) was treated with Ac.sub.2O (0.14 mL, 1.38 mM), the mixture stirred for 15 minutes then diluted with methanol (5 mL) and treated with Pd 10%/C (100 mg) under an atmosphere of H.sub.2 (balloon) over night. Nitrogen was bubbled in the mixture which was then filtered on celite using methanol, the solvent was evaporated. The residue was purified using reverse phase prep HPLC C.sub.18 column 25% CH.sub.3CN/water (0.15 HCOOH), solvent evaporated, the residue lyophilized over night, giving 50 mg of the titled compound of Ex.1 as the formate salt.

Example 2: Synthesis of Trihexamidine Isethionate (Compound #2)

(21) Compound #2 was prepared as generally presented in Example 1. Synthesis was modified to provide the proper ratio of isethionate salt, as presented below:

(22) ##STR00014##

Example 3: Synthesis of Trioctamidine Isethionate (Compound #3)

(23) ##STR00015##

(24) Step 1:

(25) To 7.2 g (60.8 mM) of cyanophenol and potassium carbonate (12.6 g, 91.2 mM) was added CH.sub.3CN (61 mL) and 1,8-dibromooctane (56 mL, 304 mM) and the mixture was refluxed over weekend. The mixture was cooled to room temperature, the solid was filtered off, washed with EtOAc, the solvent evaporated and the residue purified by combiflash to afford 15 g of A.

(26) ##STR00016##

(27) Step 2:

(28) To the triphenol (894 mg, 7.1 mM) was added DMF (30 mL), cesium carbonate (11.5 g, 35.4 mM) and the bromo A (11 g, 35.4 mM). The mixture was heated at 65° C. over night. After cooling to room temperature the mixture was diluted with water, extracted with EtOAc (2×), washed with water (4×), brine, dried with Na.sub.2SO.sub.4, filtered and the solvent evaporated, after purification on the combiflash compound B (1.32 g pure and 2.8 g impure) was obtained.

(29) ##STR00017##

(30) Step 3:

(31) To tricyano (1 g, 1.23 mM), in ethanol (6 mL) and dichloromethane (8 mL) in an ice water bath was added acetyl chloride (4.6 mL) the mixture was stoppered and stirred over weekend at room temperature. The mixture was diluted with CH.sub.2Cl.sub.2, washed with saturated NaHCO.sub.3 (2×), brine, dried over Na.sub.2SO.sub.4, filtered evaporated and purified on the combiflash, giving 962 mg of product.

(32) ##STR00018##

(33) Step 4:

(34) To 93.5 mg (0.09 mM) of starting material in ethanol (2 mL) and 7N NH.sub.3 in methanol (4 mL) was added ammonium isethionate (38.7 mg, 0.27 mM) and the mixture was heated at 65 C, cooled to room temperature, the solvent was evaporated and coevaporated with water (2×), the residue was freeze dried over night giving 96 mg of the titled compound of Example 3.

Example 4: Synthesis of Tripropamidine Formate (Compound #4)

(35) ##STR00019##

(36) Step 1:

(37) To 11.9 g (100 mM) of cyanophenol and potassium carbonate (20.7 g, 150 mM) was added CH.sub.3CN (200 mL) and 1,3-dibromopropane (50.7 mL, 500 mM) and the mixture was refluxed overnight. The mixture was cooled to room temperature, the solid was filtered off, the solvent evaporated, diluted with ether, filtered again, the solvent evaporated and the residue purified by combiflash to afford A.

(38) ##STR00020##

(39) Step 2:

(40) To the triphenol (1.05 g, 8.3 mM) was added DMF (30 mL), cesium carbonate (13.6 g, 41.6 mM) and the bromo A (10 g, 41.6 mM). The mixture was heated at 65° C. over night. After cooling to room temperature the mixture was diluted with water, extracted with EtOAc (2×), washed with water (3×), brine, dried with Na.sub.2SO.sub.4, filtered and the solvent evaporated, after purification on the combiflash compound B was obtained.

(41) ##STR00021##

(42) Step 3:

(43) A mixture of tricyano B (430 mg, 0.71 mM), hydroxylamine hydrochloride (297 mg, 4.27 mM) and triethylamine (0.59 mL, 4.27 mM) in ethanol 20 mL was refluxed over night. Upon cooling the product solidified and was filtered, washed with ethanol, air dried, giving 340 mg of C.

(44) ##STR00022##

(45) Step 4:

(46) C (340 mg, 0.48 mM) in AcOH (7 mL) was treated with Ac.sub.2O (0.21 mL, 2.18 mM), the mixture stirred for 15 minutes then diluted with methanol (5 mL) and treated with Pd 10%/C (100 mg) under an atmosphere of H.sub.2 (balloon) over night. Nitrogen was bubbled in the mixture which was then filtered on celite using methanol, the solvent was evaporated. The residue was purified using reverse phase prep HPLC C.sub.18 column 25% CH.sub.3CN/water (0.15 HCOOH), solvent evaporated, the residue lyophilized over night, giving 122 mg of the titled compound of Ex.4 as the formate salt.

Example 5: Synthesis of Triamidine Formate (Chain Average Mn 1000) (Compound #5)

(47) ##STR00023##

(48) Step 1:

(49) To a solution of glycerol ethoxylate (10 g, 10 mM) in CH.sub.2Cl.sub.2 (50 mL), THF (50 mL) and triethylamine (5.4 mL, 39 mM) in an ice/water bath was slowly added methanesulfonyl chloride (2.6 mL, 33 mM), the mixture was stirred over night at room temperature. The mixture was diluted with water, organics separated, the water reextracted with CH.sub.2Cl.sub.2, organics combined, dried with Na.sub.2SO.sub.4, filtered and the solvent evaporated. Giving 14.32 g of A.

(50) ##STR00024##

(51) Step 2:

(52) The crude from A (10 mM) was dissolved in DMF (50 mL), cesium carbonate (19.5 g, 60 mM) and 4-cyanophenol (5.96 g, 50 mM) were added and the mixture was heated over night at 60° C. After cooling to room temperature the mixture was diluted with water, extracted with EtOAc (2×), washed with water (3×), brine, dried with Na.sub.2SO.sub.4, filtered and the solvent evaporated. Purification on the combiflash afforded 11.2 g of B.

(53) ##STR00025##

(54) Step 3:

(55) A solution of B (4.4 g, 3.4 mM), hydroxylamine hydrochloride (1.42 g, 20.4 mM), triethylamine (2.84 mL, 20.4 mM) in ethanol (150 mL) was refluxed over night, the solvent was then evaporated and the residue purified by combiflash 0% to 50% methanol/CH.sub.2Cl.sub.2, giving 2.63 g of C.

(56) ##STR00026##

(57) Step 4:

(58) C (1 g, 0.71 mM) in AcOH (10 mL) was treated with Ac.sub.2O (0.33 mL, 3.46 mM), the mixture stirred for 15 minutes then diluted with methanol (5 mL) and treated with Pd 10%/C (100 mg) under an atmosphere of H.sub.2 (balloon) over night. Nitrogen was bubbled in the mixture which was then filtered on celite using methanol, the solvent was evaporated. The residue was purified using a silica gel column on the combiflash 0% to 80% methanol/CH.sub.2Cl.sub.2, methanol containing 5% HCOOH, giving 65 mg of the titled compound of Ex.5. Low resolution Mass Spectroscopy (+): 1371.9 [M+1].

Example 6: HCT Assay

(59) The compounds were evaluated in a proliferation assay using HCT-116 cells using an xCELLigence system. The results are shown in Table 2 below. Briefly, the xCELLigence system monitors cellular events in real time by measuring electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture E-Plates. The impedance measurement provides quantitative information about the biological status of the cells, including cell number, viability, and morphology. Real-time proliferation assays were then to determine the IC50 values. All proliferation assays start 24 hours after the transfection.

(60) TABLE-US-00003 TABLE 2 HCT- 116 Com- IC50 pound Structure Mw (μM) 1 Parent: 780.99 Salt: 919.07 g/mol 1.9 2 Parent: 780.99 Salt: 1159.39 g/mol 2.66 3 Parent: 865.1 g/mol Salt: 1243.55 g/mol 6.67 4 0 Parent: 654.76 Salt: 792.83 g/mol 38 5 (weight average molecular weight) Parent: 1371.61 by MS main peak Salt: 1509.68 g/mol ∞

Example 7: GalN/LPS Fulminant Liver Injury Model: An Anti-Inflammatory, Anti-TNFα, Hepatoprotection Model

(61) A Galactosamine/Liposaccharide (GalN/LPS) fulminant liver injury model may be used to evaluate the anti-inflammatory, anti-TNFα and hepatoprotective properties of the compound of the description.

(62) Mice were maintained under 12 h light/dark cycles with unlimited access to food and water. Study was performed in 6- to 14-week old male mice. C57BL/6 mice were used. Mice are first injected with pentamidine or Example 2 compound (IP injection). 30 minutes later, liver injury is induced by intraperitoneal injections of 100 μg/kg of LPS (E. coli 0111:B4) and 700 mg/kg of GalN (Sigma) dissolved in phosphate-buffered saline (PBS).

(63) Study a

(64) The tested compounds were be dosed IP 30-minutes prior to the co-treatment with galactosamine (GalN) and endotoxin (Lipopolysaccharide) LPS. Mice were either sacrificed at 6.5 hours after GalN/LPS for ALT analysis, or kept for longer periods to evaluate survival. The analysis of alanine transaminase (ALT) serum levels and histological observations allow the evaluation of the hepatoprotective, anti-TNF-α, anti-inflammatory and/or anti-fibrotic activity.

(65) Survival was evaluated at two time points, i.e. 8 and 23 hours. In controls group receiving PBS prior to the GalN/LPS liver injury, no mice survived 8 hours. In groups treated with control pentamidine at doses of 25 and 40 mg/kg, only the 40 mg/kg (67 μmole/kg) group benefited from hepatoprotective properties of pentamidine at 8 hours, but none survived 23 hours. In the case of trihexamidine isethionate, all mice survived more than 23 hours with 10 mg/kg (8.6 μmole/kg) doses. These results strongly support the hepatoprotective activity of triamidines (FIGS. 1 and 2).

(66) Study b

(67) All doses were administered IP to six mice (n=6), 30 minutes prior to the injection of the GalN and LPS, when applicable to the group. Blood samples were collected exactly 6 hours post GalN/LPS injections, or 6.5 hours after vehicle injections.

(68) TABLE-US-00004 IP Dose (mg/kg in Tx (IP) WFI) GaIN - LPS doses ALT (IU/L) Sterile WFI N/A 700 mg/kg - 5989 10 μg/kg Pentamidine 50 700 mg/kg - 306 10 μg/kg Compound 4 (C4) 5 700 mg/kg - 731 10 μg/kg Compound 5 (C5) 5 700 mg/kg - 1072 10 μg/kg Compound 5 (C5) 10 700 mg/kg - 689 10 μg/kg Compound 2 (C2) 5 700 mg/kg - 2184 10 μg/kg Compound 2 (C2) 10 700 mg/kg - 890 10 μg/kg

(69) The tested compounds were dosed IP 30 minutes prior to the co-treatment with galactosamine (GalN) and endotoxin (Lipopolysaccharide) LPS. Mice were sacrificed at 6 hours after GalN/LPS for ALT analysis. The analysis of alanine transaminase (ALT) serum levels allows the evaluation of the hepatoprotective, anti-TNF-α, anti-inflammatory and/or anti-fibrotic activity.

(70) Results indicate that Compounds 2, 4 and 5 (C2, C4, C5) are hepatoprotective in the model, as shown by low ALT activity levels following the injection of GalN/LPS. See FIG. 3. Animals treated with pentamidine at the dose of 50 mg/kg (84 μmole/kg) benefited from hepatoprotective properties of pentamidine (positive control, Penta-50 mpk). Significant hepatoprotection was also provided by lower doses: 10 mg/kg (4.3 and 8.6 μmole/kg) for C2, 5 and 10 mg/kg (3.3 and 6.6 μmole/kg) for C4 and 5 mg/kg (6.3 mole/kg) for C5. These results strongly support the hepatoprotective activity of the tested compounds (FIG. 3).

(71) Study c

(72) All doses were administered IP to six mice (n=6), 30 minutes prior to the injection of the GalN and LPS, when applicable to the group. Blood samples were collected exactly 6 hours post GalN/LPS injections, or 6.5 hours after vehicle injections.

(73) TABLE-US-00005 IP Dose (mg/kg in Tx (IP) WFI) GaIN - LPS doses ALT (IU/L) Sterile WFI N/A 700 mg/kg - 4403 10 μg/kg Compound 4 (C4) 0.625 700 mg/kg - 1755 10 μg/kg Compound 4 (C4) 1.25 700 mg/kg - 1090 10 μg/kg Compound 4 (C4) 2.5 700 mg/kg - 555 10 μg/kg

(74) The tested compounds were dosed IP 30 minutes prior to the co-treatment with galactosamine (GalN) and endotoxin (Lipopolysaccharide) LPS. Mice were sacrificed at 6 hours after GalN/LPS for ALT analysis. The analysis of alanine transaminase (ALT) serum levels allows the evaluation of the hepatoprotective, anti-TNF-α, anti-inflammatory and/or anti-fibrotic activity.

(75) Results indicate that Compound 4 (C4) provides a dose-dependent hepatoprotection in the model, as shown by low ALT activity levels following the injection of GalN/LPS. See FIG. 4. Animals treated with pentamidine at the dose of 50 mg/kg (84 μmole/kg) benefited from hepatoprotective properties of pentamidine (positive control, Penta-50 mpk). Significant hepatoprotection was also provided by C4 at 0.625 mg/kg (0.8 mole/kg), 1.25 mg/kg (1.6 mole/kg) and 2.5 mg/kg (3.2 mole/kg) for C5. These results strongly support the hepatoprotective activity of the tested compounds (FIG. 4).

Example 8

(76) DSS-Induced Acute Colitis in Mice (Compound 2 and Compound 4)

(77) Study Summary

(78) Formulation

(79) Dextran Sulfate Sodium 2.5% stocks were prepared by adding tap water in the pre-weighed powder. DSS solutions will be changed every 3 days for all groups, i.e. on Day 1 (D1) and Day 4 (D4).

(80) Formulations for Intraperitoneal Administration: Vehicle: sterile double distilled water (DDW) For Pentamidine (Penta) and Compounds 2 and 4, the compounds were dissolved in DDW and dosed at 2 mL/kg to yield the indicated doses.
In-Vivo Study

(81) Male C57BL/6 ELITE mice of ˜7-8 weeks old were used. After 5-day acclimatization period in animal facility, all mice were weighed and given tap water containing 2.5% DSS on Day 1, treatment also starts on Day 1.

(82) TABLE-US-00006 TABLE 1 Groups Group compound DSS dose Dose (mg/kg) volume route frequency duration termination 1 DDW 2.5% — 2 mL/kg IP q.d. D1-D6 D7 2 Pentamidine 2.5% 5 2 mL/kg IP q.d. D1-D6 D7 3 Compound 2 2.5% 2.5 2 mL/kg IP q.d. D1-D6 D7 4 Compound 2 2.5% 5 2 mL/kg IP q.d. D1-D6 D7 5 Compound 4 2.5% 2.5 2 mL/kg IP q.d. D1-D6 D7 6 Compound 4 2.5% 5 2 mL/kg IP q.d. D1-D6 D7
Husbandry

(83) The animal room environment is controlled (temperature 22±0.2° C.; relative humidity 55±25%; 12 hours light/dark cycle, and 12 air changes per hour). A standard certified commercial rodent chow is provided to the animals ad libitum. Procedures involving the care and use of animals in this study will be reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to conduct.

(84) Treatment and Daily Observations

(85) On the morning of Day 1 (D1), the drinking water will be replaced by 2.5% DSS-containing tap water.

(86) Animals were dosed from D1 to D6, once daily by IP injection (2 mL/kg). Animals were terminated on D7. The Disease Activity Index (DAI) scale is based on the evaluation of different parameters characterizing experimental colitis induction and progression. Body weight, presence of gross blood in the feces and stool consistency will be recorded daily.

(87) Disease Accumulated Index is Determined by Scoring Changes in:

(88) Weight loss: 0=none; 1=1 to 5%; 2=5 to 10%; 3=_10 to 20%; 4=>20% Stool consistency: 0=normal; 2=loose; 4=diarrhea Rectal bleeding: 0=normal; 2=occult bleeding; 4=gross bleeding

(89) Moreover, animals will be monitored for pain level and hydration. If they seem in distress or obtain a final Disease Activity Index of 12 points (per mouse), they will be considered reaching clinical end point for euthanasia.

(90) Termination

(91) On Day 7, mice will be weighed and disease scores evaluated, and then euthanized by CO.sub.2 inhalation.

CONCLUSION

(92) Compounds 2 and 4 have reduced the disease activity index in the DSS-induce acute colitis model, demonstrating their potential in treating GI disorders related to inflammatory conditions. The results are shown in FIG. 5.