COMPOUNDS AND THEIR USE FOR THE TREATMENT OF ALPHA1-ANTITRYPSIN DEFICIENCY

Abstract

The invention relates to specified benzamide compounds of formula (1), and pharmaceutical compositions containing the compounds. The compounds may be inducers of α.sub.1-antitrypsin (A1AT), and may be used in the treatment of a disease or disorder such as α.sub.1-antitrypsin deficiency (A1AD or AATD).

Claims

1. A compound represented by the structure: ##STR00022## ##STR00023## or pharmaceutically acceptable salt of any one thereof.

2. The compound of claim 1, wherein the compound is in a crystalline form.

3. A pharmaceutical composition comprising a compound represented by the structure: ##STR00024## ##STR00025## or pharmaceutically acceptable salt of any one thereof, and a pharmaceutically acceptable carrier.

4.-5. (canceled)

6. A method of inducing Z A1AT secretion in a subject in need thereof comprising administering to the subject a compound represented by the structure: ##STR00026## ##STR00027## ##STR00028## or pharmaceutically acceptable salt of any one thereof.

7.-11. (canceled)

12. The compound of claim 1, wherein the compound is represented by: ##STR00029## or a pharmaceutically acceptable salt of any one thereof.

13. The compound of claim 12, wherein the compound is represented by: ##STR00030## or a pharmaceutically acceptable salt of any one thereof.

14. The compound of claim 13, wherein the compound is represented by: ##STR00031## or a pharmaceutically acceptable salt thereof.

15. The compound of claim 13, wherein the compound is represented by: ##STR00032## or a pharmaceutically acceptable salt thereof.

16. The compound of claim 13, wherein the compound is represented by: ##STR00033## or a pharmaceutically acceptable salt thereof.

17. The compound of claim 13, wherein the compound is represented by: ##STR00034## or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical composition of claim 3, wherein the compound is represented by: ##STR00035## or a pharmaceutically acceptable salt of any one thereof.

19. The pharmaceutical composition of claim 18, wherein the compound is represented by ##STR00036## or pharmaceutically acceptable salt of any one thereof.

20. The method of claim 6, wherein the compound is represented by: ##STR00037## or pharmaceutically acceptable salt of any one thereof.

21. The method of claim 20, wherein the compound is represented by: ##STR00038## or pharmaceutically acceptable salt of any one thereof.

22. The method of claim 21, wherein the compound is represented by: ##STR00039## or a pharmaceutically acceptable salt thereof.

23. The method of claim 21, wherein the compound is represented by: ##STR00040## or a pharmaceutically acceptable salt thereof.

24. The method of claim 21, wherein the compound is represented by: ##STR00041## or a pharmaceutically acceptable salt thereof.

25. The method of claim 21, wherein the compound is represented by: ##STR00042## or a pharmaceutically acceptable salt thereof.

26. The method of claim 21, wherein the compound is represented by: ##STR00043## or a pharmaceutically acceptable salt thereof.

Description

EXPERIMENTAL

Example 1: N-Ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0065] N-Ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared using the following sequential synthesis procedures.

##STR00002##

Step a—Synthesis of tert-butyl 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoate

[0066] ##STR00003##

[0067] Pyrimidin-4(3H)-one (5 g, 32 mmol) and caesium carbonate (50.85 g, 156 mmol) were stirred in dimethylformamide (50 ml) for 10 minutes at room temperature. Tert-butyl 4-(bromomethyl)benzoate (14.11 g, 52 mmol) was added and the reaction was stirred for 3 hours. The reaction was diluted with water and the resulting yellow precipitate collected by filtration. The crude product was purified by column chromatography on silica, eluting with ethyl acetate/hexane (30% to 33%) to give tert-butyl 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoate. Tlc Rf 0.2 1:1 Ethyl acetate/hexane.

Step b—Synthesis of 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic acid

[0068] ##STR00004##

[0069] Tert-butyl 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoate (10 g, 35 mmol) was dissolved in dichloromethane (50 ml) and trifluoroacetic acid (70 ml) was added slowly. The reaction was stirred for 3 hours at room temperature. The reaction was concentrated under reduced pressure and the resulting oil stirred with diethyl ether (300 ml) for 20 minutes at room temperature. The resultant solid was collected by filtration, washed with diethyl ether (2×30 ml) and dried in vacuo to give 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic acid.

Step c—Synthesis of N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0070] ##STR00005##

[0071] 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic acid (64 mg, 0.27 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (86 mg, 0.54 mmol) were stirred in tetrahydrofuran (1 ml) for 10 minutes at 0° C. under nitrogen. The reaction was then allowed to warm to room temperature. Triethylamine (0.11 ml, 81 mmol) and ethylmethylamine (2M solution in tetrahydrofuran, 69 mmol) were added and the reaction was stirred for 2 hours. The reaction was concentrated under reduced pressure and the residue columned on silica eluting with 4% methanol in dichloromethane. Product containing fractions were concentrated to give N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide.

[0072] m/z: 270.96 (calc 271.13)

[0073] .sup.1H NMR (400 MHz, d6 DMSO) δ 8.69 (1H, s), 7.94 (1H, d), 7.36 (4H, s), 6.44 (1H, d), 5.13 (2H, s), 3.43 (1H, br s), 3.17 (1H, br s), 2.88 (3H, br s), 1.05 (3H, br s).

Example 2: 3-(4-(Pyrrolidine-1-carbonyl)benzyl)pyrimidin-4(3H)-one

[0074] ##STR00006##

[0075] 3-(4-(Pyrrolidine-1-carbonyl)benzyl)pyrimidin-4(3H)-one was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using pyrrolidine instead of ethylmethylamine in step c.

[0076] m/z: 282.91 (calc 283.13)

[0077] 1H NMR (400 MHz, d6 DMSO) δ 8.69 (1H, s), 7.94 (1H, d), 7.49 (2H, d), 7.35 (2H, d), 6.44 (1H, d), 5.13 (2H, s), 3.44 (2H, t), 3.34 (2H, t), 1.83 (4H, m).

Example 3: 3-(4-(Morpholine-4-carbonyl)benzyl)pyrimidin-4(3H)-one

[0078] ##STR00007##

[0079] 3-(4-(Morpholine-4-carbonyl)benzyl)pyrimidin-4(3H)-one was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using morpholine instead of ethylmethylamine in step c.

[0080] m/z: 298.97 (calc 299.13)

[0081] 1H NMR (400 MHz, d6 DMSO) δ 8.69 (1H, s), 7.94 (1H, d), 7.38 (4H, m), 6.43 (1H, d), 5.13 (2H, s), 3.59-3.34 (8H, br m).

Example 4: 3-(4-(4-Methylpiperazine-1-carbonyl)benzyl)pyrimidin-4(3H)-one

[0082] ##STR00008##

[0083] 3-(4-(4-Methylpiperazine-1-carbonyl)benzyl)pyrimidin-4(3H)-one was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using N-methylpiperazine instead of ethylmethylamine in step c.

[0084] m/z: 312.18 (calc 312.16)

[0085] 1H NMR (400 MHz, d6 DMSO) δ 8.69 (1H, s), 7.94 (1H, d), 7.36 (4H, s), 6.43 (1H, d), 5.13 (2H, s), 3.58 (2H, br), 3.33 (2H), 2.29 (4H, br m), 2.17 (3H, br s).

Example 5: N-Methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide

[0086] ##STR00009##

[0087] N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using quinazolin-4(3H)-one instead of pyrimidin-4(3H)-one in step a and methylamine instead of ethylmethylamine in step c.

[0088] m/z: 292.92 (calc 293.12)

[0089] 1H NMR (400 MHz, d6 DMSO) δ 8.60 (1H, s), 8.41 (1H, br m), 8.15 (1H, dd), 7.85 (1H, m), 7.79 (2H, d), 7.71 (1H, d), 7.56 (1H, m), 7.42 (2H, d), 5.24 (2H, s), 2.75 (3H, d).

Example 6: 4-((6-chloro-4-oxoquinazolin-3(4H)-yl)methyl)-N-methylbenzamide

[0090] ##STR00010##

[0091] 4-((6-chloro-4-oxoquinazolin-3(4H)-yl)methyl)-N-methylbenzamide was prepared similarly to N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide using 6-chloroquinazolin-4(3H)-one instead of quinazolin-4(3H)-one in step a.

[0092] m/z: 326.92, 329.10 (calc 327.08, 329.07)

[0093] 1H NMR (400 MHz, d6 DMSO) δ 8.63 (1H, s), 8.42 (1H, br m), 8.09 (1H, d), 7.88 (1H, m), 7.79 (2H, d), 7.74 (1H, d), 7.42 (2H, d), 5.24 (2H, s), 2.75 (3H, d).

Example 7: N-isopropyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0094] ##STR00011##

[0095] N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using N,N-dimethylpropan-2-amine instead of ethylmethylamine in step c.

[0096] m/z: 285.10 (calc 285.15)

[0097] 1H NMR (400 MHz, d6 DMSO) δ 8.68 (1H, s), 7.93 (1H, br m), 7.34 (4H, s), 6.43 (1H, br m), 5.12 (2H, s), 3.75 (1H br m), 2.67-2.74 (3H), 1.10 (6H).

Example 8: N-benzyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0098] ##STR00012##

[0099] N-benzyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using N-methyl benzylamine instead of ethylmethylamine in step c.

[0100] m/z: 333.13 (calc 333.15)

[0101] 1H NMR (400 MHz, d6 DMSO) δ 8.60 (1H, s), 7.93 (1H, d), 7.41 (7H, m), 7.29 (3H, m), 6.42 (1H, d), 5.16 (2H, s).

Example 9: N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)-N-(2,2,2-trifluoroethyl)benzamide

[0102] ##STR00013##

[0103] N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)-N-(2,2,2-trifluoroethyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using 2,2,2-trifluoro-N-methylethan-1-amine instead of ethylmethylamine in step c.

[0104] m/z: 325.12 (calc 325.10)

[0105] 1H NMR (400 MHz, d6 DMSO) δ 8.68 (1H, s), 7.94 (1H, d), 7.39 (4H, br s), 6.44 (1H, d), 5.13 (2H, s), 4.33 (2H, br), 3.00 (3H, br s).

Example 10: 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0106] ##STR00014##

[0107] 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using ammonium chloride instead of ethylmethylamine in step c.

[0108] m/z (M+Na): 252.07 (calc 252.07)

[0109] 1H NMR (400 MHz, d6 DMSO) δ 8.68 (1H, s), 7.95 (1H, s,), 7.94 (1H, d), 7.86-7.81 (2H, m), 7.37 (1H, s), 7.40-7.34 (2H, m), 6.43 (1H, d), 5.14 (2H, s).

Example 11: N,N-diethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0110] ##STR00015##

[0111] N,N-diethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using diethylamine instead of ethylmethylamine in step c.

[0112] m/z (M+Na): 308.14 (calc 308.14)

[0113] 1H NMR (400 MHz, d6 DMSO) δ 8.68 (1H, s), 7.94 (1H, d), 7.37-7.30 (4H, m), 6.44 (1H, d), 5.13 (2H, s), 3.48-3.37 (2H, m), 3.22-3.08 (2H, m), 1.17-1.08 (3H, m), 1.08-0.96 (3H, m).

Example 12: N-isopropyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0114] ##STR00016##

N-isopropyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using isopropylamine instead of ethylmethylamine in step c.

[0115] m/z (M+Na): 294.12 (calc 294.12)

[0116] 1H NMR (400 MHz, d6 DMSO) δ 8.63 (1H, s), 7.93 (1H, s), 7.79-7.73 (2H, m), 7.38-7.32 (2H, m), 6.43 (1H, d), 5.12 (2H, s), 4.04 (1H, sept.), 1.12 (6H, d).

Example 13: N-(4-fluorobenzyl)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0117] ##STR00017##

N-(4-fluorobenzyl)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using 4-fluorobenzylamine instead of ethylmethylamine in step c.

[0118] m/z (M+Na): 360.11 (calc 360.11)

[0119] 1H NMR (400 MHz, d6 DMSO) δ 9.03 (1H, t), 8.68 (1H, s), 7.94 (1H, d), 7.88-7.83 (2H, m), 7.42-7.38 (2H, m), 7.34 (2H, dd), 7.17-7.11 (2H, m), 6.43 (1H, d), 5.15 (2H, s), 4.44 (2H, d).

Example 14: 4-((6-oxopyrimidin-1(6H)-yl)methyl)-N-(2,2,2-trifluoroethyl)benzamide

[0120] ##STR00018##

[0121] 4-((6-oxopyrimidin-1(6H)-yl)methyl)-N-(2,2,2-trifluoroethyl)benzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using 2,2,2-trifluoroethan-1-amine instead of ethylmethylamine in step c.

[0122] m/z (M+Na): 334.08 (calc 334.08)

[0123] 1H NMR (400 MHz, d6 DMSO) δ 9.07 (1H, t), 8.69 (1H, s), 7.95 (1H, d), 7.89-7.81 (2H, m), 7.47-7.36 (2H, m), 6.44 (1H, d), 5.16 (2H, s), 4.08 (2H, qd).

Example 15: N-isopropyl-N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide

[0124] ##STR00019##

[0125] N-isopropyl-N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide was prepared similarly to N-Methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide using isopropyl methylamine in step c.

[0126] m/z: 335.28 (calc 335.16)

[0127] 1H NMR (400 MHz, d6 DMSO) δ 8.59 (1H, s), 8.15 (1H, d), 7.84 (1H, t), 7.70 (1H, d), 7.56 (1H, t), 7.39 (2H, m), 7.32 (2H, br), 5.22 (2H, s), 4.66 and 3.75 (1H, br), 2.86-2.66 (3H, br), 1.06 (6H, br).

Example 16: N-benzyl-N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide

[0128] ##STR00020##

[0129] N-benzyl-N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide was prepared similarly to N-Methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide using N-methyl benzylamine in step c.

[0130] m/z: 383.19 (calc 383.16)

[0131] 1H NMR (400 MHz, d6 DMSO) δ 8.58 (1H, d), 8.14 (1H, d), 7.83 (1H, t), 7.70 (1H, d), 7.75 (1H, t), 7.40-7.00 (9H, br), 5.23 (2H, s), 4.64 and 4.43 (2H, br), 2.84-2.78 (3H, br).

Example 17: N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)-N-(2,2,2-trifluoroethyl)benzamide

[0132] ##STR00021##

[0133] N-methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)-N-(2,2,2-trifluoroethyl)benzamide was prepared similarly to N-Methyl-4-((4-oxoquinazolin-3(4H)-yl)methyl)benzamide using 2,2,2-trifluoro-N-methylethan-1-amine in step c.

[0134] m/z: 375.19 (calc 375.12)

[0135] 1H NMR (400 MHz, d6 DMSO) δ 8.56 (1H, d), 8.12 (1H, d), 7.81 (1H, t), 7.67 (1H, d), 7.52 (1H, t), 7.39 (4H, br), 5.21 (2H, s), 4.29 and 4.10 (2H, br), 3.00 (3H, br).

Example 18: 4-((4,5-dimethyl-6-oxopyrimidin-1(6H)-yl)methyl)-N,N-dimethylbenzamide

[0136] 4-((4,5-dimethyl-6-oxopyrimidin-1(6H)-yl)methyl)-N,N-dimethylbenzamide was prepared similarly to N-ethyl-N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using 5,6-dimethylpyrimidin-4(3H)-one instead of pyrimidin-4(3H)-one in step a and dimethylamine instead of ethylmethylamine in step c.

[0137] m/z: 285.01 (calc 285.15)

[0138] 1H NMR (400 MHz, d6 DMSO) δ 8.45 (1H, s), 7.34 (4H, ABq), 5.09 (2H, s), 3.95-2.86 (3H, br), 2.20 (3H, s), 1.93 (3H, s).

Example 19: Activity of Compounds of the Invention in an A1AT Cell Secretion Assay Using HEK-Z Cells

Methods

[0139] HEK-Z cells, a human embryonic kidney cell line stably transfected with the human Z A1AT gene, were plated into 96 well plates (3.0×10.sup.5 cells/ml with 200 μl of media/well) overnight at 37° C. in a humidified atmosphere containing 5% CO.sub.2. Following incubation cells were washed with 200 μl serum-free media three times and media was replaced with treatments in quadruplicate using serum free media containing either vehicle, 10 μM suberanilohydroxamic acid (SAHA) or a compound of the invention (at concentrations of 10, 33, 100 and 333 nM) for 48 h in a 37° C. incubator in a final volume of 200 μl. At the end of the incubation step the supernatants were removed from the wells, centrifuged at 1000×g at 4° C. for 10 min and were assayed for human A1AT levels by ELISA (Human Serpin A1/α.sub.1-antitrypsin duo set ELISA, R& D Systems, DY1268) per manufacturer's instructions.

[0140] Briefly, a 96 well plate was coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume/well). The capture antibody was then removed and wells washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 h at room temperature. Diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to each well in duplicate and the plates were covered with a plate sealer and left at room temperature for 2 h. At the end of the sample incubation step, samples were removed and all wells washed as previously and 100 μl detection antibody (1:180 dilution from stock) was added to each well and incubated for a further 2 h at room temperature. Following incubation with detection antibody, supernatant was removed and wells were washed as previously and 100 μl streptavidin-HRP solution (1:200 dilution from stock) was added to each well for 20 min in the dark. After which, 50 μl stop solution (2M H.sub.2SO.sub.4) was added and optical density (OD) of each well was read at 450 nm with 570 nm blank subtracted from each well using a microplate reader. A 4 parameter logistic curve was constructed using GraphPad Prism 7 and A1AT concentrations were determined in each sample by interpolation from a standard curve and multiplying by the appropriate dilution factor.

Results

[0141] The data in Table 1 show that compounds of Examples 1-18 increase secretion of Z A1AT from HEK-Z cells at 300 nM.

TABLE-US-00001 TABLE 1 Median A1AT % increase Example over vehicle at 300 nM 1 330 2 350 3 280 4 390 5 180 6 200 7 160 8 150 9 170 10 260 11 230 12 210 13 190 14 180 15 190 16 160 17 170 18 170