Compounds and their use in treating alpha 1-antitrypsin deficiency

Abstract

The present invention relates to oxopyrimidinyl-methyl-benzamide derivatives, such as in the form of pharmaceutically acceptable salts or crystals, pharmaceutical compositions comprising the derivatives, and medical uses thereof, in particular for the treatment of alpha 1-antitrypsin deficiency (A1AD or AATD).

Description

[0001] The present invention relates to N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide and related compounds and their medical uses.

[0002] α1-antitrypsin (A1AT) is a member of the serpin superfamily produced by the liver and secreted into the blood. It inhibits a variety of serine proteases, especially neutrophil elastase. When A1AT levels in the blood are low, excessive neutrophil elastase activity can degrade lung tissue, leading to respiratory complications such as chronic obstructive pulmonary disease (COPD).

[0003] The reference range for A1AT in the blood is 0.9-2.3 g / L. Levels below this are typical of alpha 1-antitrypsin deficiency (A1AD or AATD), a genetic disease caused by mutations in the SERPINA1 gene encoding A1AT. The Z mutation, the most common cause of AATD, is a glutamic acid replaced by lysine at position 366 of A1AT (UniProtKB-P01009 (A1AT_HUMAN)), corresponding to the replacement of glutamic acid at position 342 of the mature protein (Z A1AT). The Z mutation affects the folding of A1AT, resulting in only a small fraction acquiring the native / active state. The rest is either cleared as misfolded protein or accumulates in the liver as stable aggregates. Due to misfolding, homozygous carriers of the Z mutation (ZZ) have plasma A1AT levels that are 10-15% of normal, making carriers susceptible to COPD. The accumulation of Z A1AT aggregates in hepatocytes makes carriers susceptible to cirrhosis, liver cancer, and other liver diseases.

[0004] Current treatment for the pulmonary manifestations of AATD includes augmented therapy using A1AT concentrates prepared from plasma from blood donors. Four A1AT products have been approved for use by the U.S. FDA: Prolastin, Zemaira, Glassia, and Aralast. Administration is by intravenous infusion once weekly. Augmented therapy has been shown to slow the progression of COPD. Hepatic manifestations of AATD (e.g., cirrhosis and cancer) are treated with steroids and liver transplantation. Investigational approaches to improve treatment of hepatic manifestations include inhibiting ZA1AT aggregation and increasing the clearance of aggregates by activating autophagy. Investigational approaches to improve treatment of both pulmonary and hepatic manifestations are aimed at improving ZA1AT folding and secretion.

[0005] Elliott et al. (Protein Science, 2000, 9, 1274-1281) described the X-ray crystal structure of A1AT and identified five cavities as potential targets for rational drug design to develop drugs that would affect Z A1AT polymerization.

[0006] Parfrey et al. (J. Biol. Chem., 2003, 278, 35, 33060-33066) further defined a cavity as a potential target for rational drug design to develop drugs that would affect Z A1AT polymerization.

[0007] Knaupp et al. (J. Mol. Biol., 2010, 396, 375–383) showed that bis-ANS (4,4'-diphenylamino-1,1'-binaphthyl-5,5'-disulfonate) can be used in a 1:1 stoichiometric ratio and a K of 700 nM. d Binds to Z A1AT but not to wild-type A1AT(M).

[0008] Chang et al. (J. Cell. Mol. Med., 2009, 13, 8B, 2304-2316) reported a series of peptides that inhibit Z A1AT polymerization, including Ac-TTAI-NH2.

[0009] Burrows et al. (Proc. Nat. Acad. Sci., 2000, 97, 4, 1796-1801) showed that a series of non-selective molecular chaperones (including 4-phenylbutyrate, glycerol and trimethylamine oxide) were able to increase ZA1AT levels in cell supernatants and mouse models.

[0010] Bouchecareilh et al. (Journal of Biological Chemistry, 2012, 287, 45, 38265-38278) describe the use of histone deacetylase inhibitors, in particular SAHA (suberoylanilide hydroxamic acid), to increase the secretion of wild-type (M) and Z A1AT by cells.

[0011] Berthelier et al. (PLOS ONE, May 11, 2015) have demonstrated that S-(4-nitrobenzyl)-6-thioguanine can prevent Z A1AT polymerization in vitro.

[0012] Mallya et al. (J. Med. Chem., 2007, 50, 22, 5357-5363) described a series of phenols that were able to block Z A1AT polymerization in vitro, such as N-(4-hydroxy-3,5-dimethylphenyl)-2,5-dimethylthiophene-3-sulfonamide.

[0013] Huntington (13th International Symposium on Proteases, Inhibitors and Biocontrol, September 23, 2012, and 7th International Symposium on Serpin Biology, Structure and Function, April 1, 2014) discussed the cavity from the X-ray crystal structure of ZA1AT as a potential target for rational drug design to develop drugs that will affect ZA1AT polymerization.

[0014] US Pat. No. 8,436,013 B2 discloses various structures capable of increasing Z A1AT secretion from cells in the micromolar range.

[0015] Compounds with CAS Registry Numbers 1797054-78-4 and 1219580-65-0 are listed in the Aurora Building Blocks catalog.

[0016] According to one aspect of the present invention, there is provided N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

[0017]

[0018] We found that N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide surprisingly showed very effective in increasing the levels of correctly folded and therefore active Z A1AT while having no effect on the secretion of wild-type (M) A1AT or the Siiyama variant of A1AT.

[0019] According to another aspect of the present invention, there is provided N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

[0020]

[0021] We found that N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide surprisingly showed very effective in increasing the levels of correctly folded and therefore active Z A1AT while having no effect on the secretion of wild-type (M) A1AT or the Siiyama variant of A1AT.

[0022] According to another aspect of the present invention, there is provided N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

[0023]

[0024] We found that N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide surprisingly showed very effective in increasing the levels of correctly folded and therefore active Z A1AT while having no effect on the secretion of wild-type (M) A1AT or the Siiyama variant of A1AT.

[0025] The compound of the present invention may be in the form of a pharmaceutically acceptable salt or a crystal.

[0026] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable mono-organic salt or inorganic salt of the compounds of this invention. This may include the addition salt of an inorganic acid, such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, diphosphate and nitrate, or the addition salt of an organic acid, such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, mesylate, p-toluenesulfonate, palmitate and stearate. Exemplary salts also include oxalate, chloride, bromide, iodide, bisulfate, acid phosphate, isonicotinate, salicylate, acid citrate, oleate, tannate, pantothenate, bitartrate, ascorbate, gentisate, gluconate, glucuronate, sucrose acid salt, formate, benzoate, glutamate, ethanesulfonate and benzenesulfonate. Other examples of pharmaceutically acceptable salts can refer to Gould (1986, Int J Pharm 33: 201-217).

[0027] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of the present invention as described herein and a pharmaceutically or therapeutically acceptable excipient or carrier.

[0028] The term "pharmaceutically or therapeutically acceptable excipient or carrier" refers to a solid or liquid filler, diluent or encapsulating material that does not interfere with the effectiveness or biological activity of the active ingredient and is non-toxic to the host (which may be a human or an animal) to which it is administered. Depending on the specific route of administration, a variety of pharmaceutically acceptable carriers, such as those known in the art, may be used. Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

[0029] All suitable modes of administration are contemplated according to the present invention. For example, the administration of the drug can be by oral, subcutaneous, direct intravenous, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient-controlled analgesia (PCA and PCEA), intramuscular, intrathecal, epidural, intracisternal, intraperitoneal, transdermal, topical, transmucosal, buccal, sublingual, transmucosal, inhalation, intranasal, intraatrial, intranasal, rectal or intraocular route. The drug can be prepared in discrete dosage units and can be prepared by any method known to the pharmaceutical field.

[0030] All suitable pharmaceutical dosage forms are contemplated. Administration of the drug can be in the form of, for example, oral solutions and suspensions, tablets, capsules, lozenges, effervescent tablets, transmucosal films, suppositories, buccal products, oral mucosal retention products, topical creams, ointments, gels, films and patches, transdermal patches, abuse-resistant and abuse-resistant formulations, sterile solutions suspensions and reservoirs for parenteral use, etc., administered with immediate release, sustained release, delayed release, controlled release, extended release, etc.

[0031] Another aspect of the invention is the use of a compound of the invention as defined herein in the manufacture of a medicament for the treatment of a disease or condition.

[0032] Another aspect of the invention is a compound of the invention for use as an inducer of ZA1AT secretion.

[0033] There is further provided a compound of the invention as defined herein for use in treating a disease or condition.

[0034] The present invention also encompasses a method of treating a disease or condition comprising the step of administering to a patient in need thereof a compound or pharmaceutical composition of the present invention as defined herein.

[0035] The present invention also includes the use of the compounds of the present invention as inducers of Z A1AT secretion. The use may be for treating a disease or condition. The use may be in vitro, for example in an in vitro assay.

[0036] A disease or condition suitable for treatment according to related aspects of the invention is one characterized by low plasma A1AT levels, such as AATD.

[0037] Numerical ranges used in this specification are intended to explicitly include all single integers within the range and all combinations of upper and lower numerical limits in the broadest range of a given range within the scope of the present invention.

[0038] As used herein, the term "comprising" should be understood to mean both comprising and consisting of. Thus, when the present invention refers to a "pharmaceutical composition comprising a compound as an active ingredient", the term is intended to encompass compositions in which other active ingredients may be present as well as compositions consisting of only one active ingredient as defined.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary experts in the field to which the invention belongs. Similarly, all publications, patent applications, all patents and all other references mentioned herein are incorporated by reference in their entirety (where permitted by law).

[0040] Specific non-limiting embodiments of the present invention will now be described with reference to the following drawings, in which:

[0041] Figure 1 is a graph showing the dose-dependent effect of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in an in vitro A1AT cellular secretion assay using HEK-EBNA cells containing the Z A1AT plasmid. Vehicle and 10 μM SAHA were tested as controls on each plate. The x-axis shows the various treatments of cells: vehicle, SAHA, and increasing concentrations of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, and the y-axis is the concentration of A1AT in the cell supernatant (ng / ml);

[0042] Figure 2 is a graph showing the effect of 10 μM N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in an in vitro A1AT cellular secretion assay using HEK-EBNA cells containing a MA1AT plasmid. Vehicle and 10 μM SAHA were tested as controls. The x-axis shows the various treatments of cells: vehicle, SAHA, and N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, and the y-axis is the concentration of A1AT in the cell supernatant (ng / ml);

[0043] Figure 3 is a graph showing the effect of 1 and 10 μM N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in an in vitro A1AT cellular secretion assay using HEK-EBNA cells containing Siiyama A1AT plasmid. Vehicle and 10 μM SAHA were tested as controls. The x-axis shows the various treatments of cells: vehicle, SAHA, and two concentrations of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, and the y-axis is the concentration of A1AT in the cell supernatant (ng / ml);

[0044] Figure 4 is a graph showing the effect of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide on Z A1AT levels in mice expressing human Z A1AT (huZ mice). Mice were treated with vehicle, 5, 15 and 50 mg / kg N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide by oral gavage twice daily for 14 consecutive days. Blood was collected and plasma was prepared on days -12, -7 and -5 to determine the circulating basal level of human Z A1AT. Plasma samples collected on the last three days of the study (days 12, 13 and 14) were used to determine the effect of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide treatment on circulating human Z A1AT levels compared to basal levels. The x-axis is the treatment dose of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in mg / kg; the y-axis is the mean percentage level of human Z A1AT in each treatment group compared to the baseline level;

[0045] Figure 5 is a graph showing the dose-dependent effect of N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in an in vitro A1AT cellular secretion assay using HEK-EBNA cells containing the Z A1AT plasmid. Vehicle and 10 μM SAHA were tested as controls on each plate. The x-axis shows the various treatments of cells: vehicle, SAHA, and increasing concentrations of N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, and the y-axis is the concentration of A1AT in the cell supernatant (ng / ml);

[0046] Figure 6 is a graph showing the dose-dependent effect of N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in an in vitro A1AT cellular secretion assay using HEK-EBNA cells containing a Z A1AT plasmid. Vehicle and 10 μM SAHA were tested on each plate as controls. The x-axis shows the various treatments of cells: vehicle, SAHA, and increasing concentrations of N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, and the y-axis is the concentration of A1AT in the cell supernatant (ng / ml); and

[0047] Fig. 7A and 7BWestern blots of Z A1AT after isolation and treatment with trypsin are shown. Z A1AT was isolated after one wild-type (15.5b) and three HuZ mice (22.1e, 24.1a, 24.1b) were treated with N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide by oral gavage at a dose level of 200 mgKg for 10 days. Each Western blot is labeled with the mouse identifier. The gel lanes are labeled according to Table 4. The protein bands are labeled "C" - complex, "NA" - native antitrypsin and "CA" - cleaved antitrypsin.

[0048] experiment

[0049] Example 1 :N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0050] N,N-Dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared using the following sequential synthetic procedure.

[0051] (a) Synthesis of tert-butyl 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoate

[0052]

[0053] At room temperature, pyrimidine-4 (3H) -one (5g, 32mmol) and cesium carbonate (50.85g, 156mmol) were stirred in dimethylformamide (50ml) for 10 minutes. 4- (bromomethyl) tert-butyl benzoate (14.11g, 52mmol) was added and the reaction was stirred for 3 hours. The reaction was diluted with water and the resulting yellow precipitate was collected by filtration. The crude product was purified by silica gel column chromatography and eluted with ethyl acetate / hexane (30% to 33%) to give tert-butyl 4- ((6-oxopyrimidine -1 (6H) -yl) methyl) benzoate. Tlc Rf 0.2 1: 1 ethyl acetate / hexane.

[0054] (b) Synthesis of 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic acid

[0055]

[0056] Tert-butyl 4-((6-oxopyrimidine-1(6H)-yl)methyl)benzoate (10 g, 35 mmol) was dissolved in dichloromethane (50 ml) and trifluoroacetic acid (70 ml) was slowly added. The reaction was stirred at room temperature for 3 hours. The reaction was concentrated under reduced pressure and the resulting oil was stirred at room temperature with ether (300 ml) for 20 minutes. The resulting solid was collected by filtration, washed with ether (2x30 ml) and dried in vacuo to give 4-((6-oxopyrimidine-1(6H)-yl)methyl)benzoic acid.

[0057] (c) Synthesis of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0058]

[0059] 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic acid (16 g, 69 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (18 g, 139 mmol) were stirred in tetrahydrofuran (100 ml) under nitrogen at 0°C for 10 minutes. The reaction was then allowed to warm to room temperature. Triethylamine (21.11 g, 208 mmol) and dimethylamine (2M in tetrahydrofuran, 69 mmol) were added and the reaction was stirred for 2 hours. The reaction was concentrated under reduced pressure and the residue was loaded on silica, eluting with 4% methanol in dichloromethane. The fractions containing the product were concentrated to give N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide.

[0060] Tlc Rf 0.8 10% methanol in dichloromethane.

[0061] m / z: 257.96 (calculated 258.12)

[0062] 1 H NMR (400MHz, d6 DMSO) δ8.69(1H,s),7.94(1H,d),7.38(4H,m),6.44(1H,d),5.13(2H,s),2.96(3H,br s),2.87(3H,br s).

[0063] N,N-Dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide can also be prepared in a similar manner to step a) by alkylation of pyrimidin-4(3H)-one with 4-(bromomethyl)-N,N-dimethylbenzamide.

[0064]

[0065] Example 2 :N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0066]

[0067] N-Methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared analogously to N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using methylamine instead of dimethylamine in step c.

[0068] m / z: 242.90 (calculated 243.10)

[0069] 1H NMR (400MHz, d6 DMSO) δ8.69(1H,s),8.42(1H,br m),7.94(1H,d),7.79(2H,d),7.37(2H,d),6.43(1H,d),5.13(2H,s),2.77(3H,s).

[0070] Example 3 :N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0071]

[0072] N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was prepared analogously to N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide using d6-dimethylamine instead of dimethylamine in step c.

[0073] Tlc Rf 0.8 10% methanol in dichloromethane.

[0074] 1 H NMR (400MHz, d6 DMSO) δ8.69(1H,s),7.94(1H,d),7.38(4H,m),6.44(1H,d),5.13(2H,s).

[0075] Example 4 : Activity of compounds of the invention in the A1AT cell secretion assay using HEK-Z cells

[0076] method

[0077] HEK-Z cells, a human embryonic kidney cell line stably transfected with human Z A1AT gene, were plated in 96-well plates (3.0×10 5cells / ml, 200 μl medium / well) and incubated at 37°C in a humidified atmosphere containing 5% CO2 overnight. After incubation, cells were washed three times with 200 μl of serum-free medium, the medium was replaced, and serum-free medium containing vehicle, 10 μM suberoylanilide hydroxamic acid (SAHA), N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide (at concentrations of 10, 33, 100, and 333 nM), N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide (at concentrations of 10, 33, 100, and 333 nM), or N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide (at concentrations of 10, 33, 100, and 333 nM) was used in quadruplicate in a final volume of 200 μl in a 37°C incubator for 48 hours. At the end of the incubation step, supernatants were removed from the wells, centrifuged at 1000 x g for 10 min at 4°C, and human A1AT levels were determined by ELISA (Human Serpin A1 / α1-antitrypsin Dual Recombinant ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0078] 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 the wells were washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl of reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 hour at room temperature. The diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg / ml A1AT) or blanks were then added to each well in duplicate, the plates were covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, the samples were removed and all wells were washed as before, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well and incubated for another 2 hours at room temperature. After incubation with the detection antibody, the supernatant was removed and the wells were washed as before, then 100 μl of streptavidin-HRP solution (diluted 1:200 from stock solution) was added to each well and kept in the dark for 20 min. After that, 50 μl of stop solution (2M H2SO4) was added and the optical density (OD) of each well was read at 450 nm using a microplate reader, and a 570 nm blank was subtracted from each well. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration in each sample was determined by interpolating from the standard curve and multiplying by the appropriate dilution factor.

[0079] result

[0080] The amount of A1AT secreted into the culture medium from transfected HEK-EBNA cells was measured by ELISA. 10 μM SAHA was used as a positive control for all in vitro A1AT secretion experiments.

[0081] Figure 1 The data in show that N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide stimulates the secretion of Z A1AT in a dose-dependent manner as measured by ELISA.

[0082] Figure 5 The data in indicate that N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide stimulates the secretion of Z A1AT in a dose-dependent manner as measured by ELISA.

[0083] Figure 6 The data in show that N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide stimulates the secretion of Z A1AT in a dose-dependent manner as measured by ELISA.

[0084] Example 5 : Activity of compounds of the invention in the A1AT cell secretion assay using HEK-M cells

[0085] method

[0086] HEK-M cells, a human embryonic kidney cell line, stably transfected with the human MA1AT gene were plated in 96-well plates (3.0×10 5 Cells / ml, 200 μl culture medium / well), in a humidified atmosphere containing 5% CO2 at 37°C overnight. After incubation, the cells were washed 3 times with 200 μl serum-free medium, and the medium was replaced with serum-free medium containing vehicle, 10 μM suberoylanilide hydroxamic acid (SAHA) or a compound of the present invention (10 μM) in six replicates with a final volume of 200 μl in a 37°C incubator for 48 hours. At the end of the incubation step, the supernatant was removed from the wells, centrifuged at 1000 x g for 10 minutes at 4°C, and human A1AT levels were determined by ELISA (human serine protease inhibitor A1 / α1-antitrypsin dual recombinant ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0087] 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 the wells were washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl of reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 hour at room temperature. The diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg / ml A1AT) or blanks were then added to each well in duplicate, the plates were covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, the samples were removed and all wells were washed as before, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well and incubated for another 2 hours at room temperature. After incubation with the detection antibody, the supernatant was removed and the wells were washed as before, then 100 μl of streptavidin-HRP solution (diluted 1:200 from stock solution) was added to each well and kept in the dark for 20 min. After that, 50 μl of stop solution (2M H2SO4) was added and the optical density (OD) of each well was read at 450 nm using a microplate reader, and a 570 nm blank was subtracted from each well. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration in each sample was determined by interpolating from the standard curve and multiplying by the appropriate dilution factor.

[0088] result

[0089] The amount of A1AT secreted into the culture medium from transfected HEK-EBNA cells was measured by ELISA. 10 μM SAHA was used as a positive control for all in vitro A1AT secretion experiments.

[0090] Figure 2 The data in indicate that N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at 10 μM does not stimulate the secretion of MA1AT as measured by ELISA. In contrast, the positive control 10 μM SAHA stimulated an increase in MA1AT secretion.

[0091] Example 6 : Activity of compounds of the invention in the A1AT cell secretion assay using HEK-Siiyama cells

[0092] The rare Siiyama mutation (Ser 53 to Phe, numbering of mature A1AT) was identified in Japanese males with AATD (Seyama et al. J Biol Chem (1991) 266: 12627-32). Ser53 is one of the conserved serpin residues and is thought to be important for the organization of the inner core of the A1AT molecule. The change from an uncharged polar amino acid to a large nonpolar amino acid on the conserved backbone of the protein affects the folding and intracellular processing of Siiyama A1AT.

[0093] method

[0094] HEK-Siiyama cells, a human embryonic kidney cell line stably transfected with the human Siiyama A1AT gene, were plated in 96-well plates (3.0×10 5 Cells / ml, 200 μl culture medium / well), in a humidified atmosphere containing 5% CO2 at 37°C overnight. After incubation, the cells were washed 3 times with 200 μl serum-free medium, and the medium was replaced with serum-free medium containing vehicle, 10 μM suberoylanilide hydroxamic acid (SAHA) or a compound of the present invention (1 or 10 μM), and treated in eight replicates with a final volume of 200 μl in a 37°C incubator for 48 hours. At the end of the incubation step, the supernatant was removed from the wells, centrifuged at 1000 x g for 10 minutes at 4°C, and human A1AT levels were determined by ELISA (human serine protease inhibitor A1 / α1-antitrypsin dual recombinant ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0095] 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 the wells were washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl of reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 hour at room temperature. The diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg / ml A1AT) or blanks were then added to each well in duplicate, the plates were covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, the samples were removed and all wells were washed as before, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well and incubated for another 2 hours at room temperature. After incubation with the detection antibody, the supernatant was removed and the wells were washed as before, then 100 μl of streptavidin-HRP solution (diluted 1:200 from stock solution) was added to each well and kept in the dark for 20 min. After that, 50 μl of stop solution (2M H2SO4) was added and the optical density (OD) of each well was read at 450 nm using a microplate reader, and a 570 nm blank was subtracted from each well. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration in each sample was determined by interpolating from the standard curve and multiplying by the appropriate dilution factor.

[0096] result

[0097] The amount of human A1AT secreted into the culture medium from transfected HEK-EBNA cells was measured by ELISA. SAHA at 10 μM was used as a positive control for all in vitro A1AT secretion experiments.

[0098] Figure 3 The data in show that N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide did not stimulate the secretion of Siiyama A1AT at 1 or 10 μM as measured by ELISA. In contrast, the positive control 10 μM SAHA stimulated an increase in Siiyama A1AT secretion.

[0099] Example 7 : Activity of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in huZ mice

[0100] The huZ mouse (also known as the PiZZ mouse) is a transgenic mouse strain that contains multiple copies of the Z variant of the human A1AT gene, developed by two independent groups, Dycaico et al. (Science (1988) 242: 1409-12) and Carlson et al. (J. Clin Invest (1989) 83: 1183-90). The PiZZ mouse is on a C57Bl / 6 background and expresses human Z A1AT protein in liver tissue. The mice used in this study were from the descendants of Carlson and his colleagues (transgenic strain Z11.03). The HuZ mouse has been used as a tool to evaluate the effects of compounds on increasing circulating levels of Z A1AT in plasma or the effects of compounds on the accumulation of Z A1AT polymers in the liver and related liver pathology.

[0101] method

[0102] HuZ mice (n=4 / group; male or female) with basal human A1AT plasma levels between 200-600 μg / ml were treated with vehicle or 5, 15 or 50 mg / kg of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide by oral gavage twice daily for 14 consecutive days. Mice had free access to food (standard mouse chow, safe diet) and water. On study day 14, each mouse was dosed one hour before the terminal procedure. Blood was collected from the tail vein of each mouse on days -12, -7 and -5 before dosing and on days 12, 13 and 14 of dosing. Blood was collected into a microdish containing EDTA, and plasma was prepared by centrifugation at 2700 x g for 10 minutes at 4°C. Aliquots of plasma were taken and stored at -80°C for bioanalysis. Plasma samples from days -12, -7 and -5 before dosing were used to determine the average basal level of human Z A1AT for each mouse. Plasma samples collected on the last three days of dosing (days 12, 13 and 14) of the study were used to determine the effect of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide on Z A1AT secretion by measuring human Z A1AT levels and comparing them with the basal levels of each mouse. Human Z A1AT levels in mouse plasma samples were measured by ELISA (human serpin A1 / α1-antitrypsin duo recombinant ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0103] 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 the wells were washed three times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200 μl of reagent diluent (25% Tween 20 in PBS) was incubated in each well for 1 hour at room temperature. The diluted samples, standards (125, 250, 500, 1000, 2000, 4000 and 8000 pg / ml A1AT) or blanks were then added to each well in duplicate, the plates were covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, the samples were removed and all wells were washed as before, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well and incubated for another 2 hours at room temperature. After incubation with the detection antibody, the supernatant was removed and the wells were washed as before, then 100 μl of streptavidin-HRP solution (diluted 1:200 from stock solution) was added to each well and kept in the dark for 20 min. After that, 50 μl of stop solution (2M H2SO4) was added and the optical density (OD) of each well was read at 450 nm using a microplate reader, and a 570 nm blank was subtracted from each well. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration in each sample was determined by interpolating from the standard curve and multiplying by the appropriate dilution factor.

[0104] result

[0105] The effect of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide on circulating levels of human ZA1AT was evaluated in the huZ mouse model. Mice were treated with 5, 15 or 50 mg / kg twice daily by oral gavage for 14 consecutive days.

[0106] Figure 4 The data in show that N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide stimulates secretion of human Z A1AT in a dose-dependent manner compared to baseline levels in huZ mice.

[0107] Example 8 :Pharmacokinetics of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in mice

[0108] N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was administered intravenously (2 mg / kg) or orally by gavage (10 mg / kg) to male C57BI / 6 mice. Whole blood diluted with water was prepared from these dosed animals over a period of time up to 24 hours after dosing to allow blood concentrations of the drug to be measured by UPLC-MS / MS. The measured drug levels allowed the calculation of the following parameters for N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

[0109] Half-life in blood (t 1 / 2 )=1.9h

[0110] Observed clearance = 7.8 ml / min / kg

[0111] Distribution volume (Vz) = 1.3 l / kg

[0112] Oral C max =9311ng / ml

[0113] AUC all =15551ng.h / ml

[0114] AUC INF =15564ng.h / ml

[0115] Oral bioavailability (F, AUC INF )=73%

[0116] Example 9 : Pharmacokinetics of the compounds of the present invention in rats

[0117] Compounds were administered intravenously (2 mg / kg) or orally by gavage (10 mg / kg) to Sprague Dawley rats. Whole blood diluted with water was prepared from these dosed animals over a period of time up to 24 hours post-dose to allow measurement of blood concentrations of the drug by UPLC-MS / MS.

[0118] The compound levels measured after administration of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide allowed the calculation of the following parameters for N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

[0119] Half-life in blood (t 1 / 2 )=55min

[0120] Observed clearance = 10.6 ml / min / kg

[0121] Distribution volume (Vz) = 0.84 l / kg

[0122] Oral C max =5326ng / ml

[0123] Oral AUC INF =16465ng.h / ml

[0124] Oral bioavailability (F) = 104%

[0125] The plasma levels of N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at various time points after a 10 mg / Kg oral dose of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide are shown in Table 1.

[0126] Table 1. Plasma Levels of N-Methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide Following Oral Dose of N,N-Dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0127]

[0128] The plasma levels of N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at various time points after a 2 mg / Kg iv dose of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide are shown in Table 2

[0129] Table 2. Plasma Levels of N-Methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide Following an Intravenous Dose of N,N-Dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0130]

[0131] The compound levels measured after administration of N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide allowed the calculation of the following parameters for N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

[0132] Half-life in blood (t 1 / 2 )=63min

[0133] Observed clearance = 10.6 ml / min / kg

[0134] Volume of distribution (Vz) = 1.0 l / kg.

[0135] The compound levels measured after administration of N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide allowed the calculation of the following parameters for N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

[0136] Half-life in blood (t 1 / 2 )=52min

[0137] Observed clearance = 10.6 ml / min / kg

[0138] Volume of distribution (Vz) = 0.72 l / kg.

[0139] Example 10 : Stability of the compounds of the present invention in mouse, rat and human hepatocytes

[0140] The intrinsic clearance (CL) of the compounds of the invention was measured in hepatocyte suspensions of cryopreserved male C57BL6 mouse hepatocytes, cryopreserved rat hepatocytes, or mixed hepatocyte suspensions of cryopreserved human hepatocytes. int ) and half-life. Briefly, compounds were incubated with hepatocyte suspensions at 37°C for a period of time, and the remaining compound at each time point was assessed by mass spectrometry (UPLC-MS / MS). CL of all compounds in mouse, rat, and human hepatocytes int All <3μl / min / 10 6 All compounds had half-lives >460 min in mouse, rat and human hepatocytes.

[0141] Embodiment 11 : Plasma protein binding of the compounds of the present invention

[0142] The extent to which the compounds of the invention bind to plasma proteins such as albumin and alpha-1 acid glycoprotein in human, rat or mouse plasma was determined by rapid equilibrium dialysis. The compounds were incubated at 5 μM for 4 hours at 37° C. The plasma protein binding of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was 37.3% in mouse plasma, 14.8% in rat plasma, and 30.6% in human plasma.

[0143] Example 12 :Activity of the compounds of the present invention against cytochrome P450

[0144] The inhibition of individual CYPs by compounds of the invention was assessed using the method described in Weaver et al., 2003, Drug Metab Dispos 31:7, 955-966 using E. coli CYPEX membranes in combination with specific probe substrates. The results are shown in Table 3.

[0145] Table 3. In vitro CYP inhibition data for N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

[0146]

[0147] Example 13 : The activity of the compounds of the present invention against HERG channels

[0148] The compounds of the invention were tested for inhibition of cardiac potassium (hERG) channels using Patchliner automated patch clamp. Six-point concentration-response curves were generated using semi-log serial dilutions from a maximum final test concentration of 100 μM. IC was obtained from a 4-parameter logistic fit of the concentration-response data. 50 N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide exhibited an IC50>100 μM, with 7% inhibition at 100 μM. The reference compound values ​​are consistent with those provided in the literature (Elkins et al., 2013 J. Pharm. Tox. Meth. 68: 11-122).

[0149] Embodiment 14 The compounds of the present invention are active against a group of enzymes, ion channels and receptors

[0150] For DiscoverX Safety47 TM The group tested compounds of the present invention. N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide showed an exceptionally clean off-target curve at 10 μM. At this concentration, no target was inhibited by more than 25%.

[0151] Embodiment 15: Water Solubility of the Compounds of the Invention

[0152] The compound of the invention was shaken with increasing amounts of water for 5 minutes until a clear solution was formed. Using this method, the solubility of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was greater than 100 mg / ml.

[0153] Example 16 : Activity of the compounds of the invention in bacterial reverse mutation assay

[0154] The purpose of the bacterial reverse mutation test is to evaluate the mutagenic potential of the test item by the ability of a specific bacterial strain to revert from auxotrophic growth to prototrophy. The assay detects point (gene) mutations at specific histidine or tryptophan loci that may be induced by compounds that cause base pair substitutions or frameshift mutations in the genomes of these organisms. The analysis is performed in the presence and absence of an exogenous mammalian oxidative metabolic system (S-9mix, a post-mitochondrial fraction of liver derived from rat livers treated with Aroclor 1254) to simulate mammalian metabolism. The test method is based on established procedures for bacterial mutagenicity testing.

[0155] N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was tested in all strains in the presence and absence of S-p9 mix up to the guideline regulatory maximum dose level of 5000 μg / plate.

[0156] There was no evidence of toxicity and no precipitation.

[0157] There was no increase in the number of revertants greater than a defined fold increase (twice the relevant negative control value for TA98, TA100 or WP2 uvrA or three times the relevant negative control value for TA1535 or TA1537) in any strain at any dose level of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide under plate introduction conditions in the presence or absence of S-9mix.

[0158] N,N-Dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was not mutagenic under the conditions of this test.

[0159] Embodiment 17 : Activity of compounds of the invention in an in vitro micronucleus assay

[0160] The purpose of the in vitro micronucleus assay is to evaluate the ability of the test item to cause chromosome or spindle damage or interfere with the cell cycle in the cytoplasm of TK6 cells in a manner that results in the formation of micronuclei. Chromosomal defects are believed to underlie many human genetic diseases. Micronuclei (MN) are the result of chromosome fragments or entire chromosomes remaining in the cytoplasm and not being integrated into the nucleus of newly formed cells. These are due to: chromosome damage resulting in acentromere fragments that cannot attach to the mitotic spindle; spindle damage that prevents chromosome attachment to the spindle or otherwise interferes with cell division. The assay is performed in the presence and absence of an exogenous mammalian oxidative metabolic system (S-9mix, a post-mitochondrial fraction of liver derived from rat liver treated with Aroclor 1254) to mimic mammalian metabolism. Cells are exposed to the test item for 3 hours and samples are taken 44 hours after the start of treatment. As many chemicals are reported to exert their active effects only after prolonged treatment, a continuous treatment period of 27 hours in the absence of S-9mix is ​​also included. This corresponds to 1.5 to 2.0 times the average generation time of the cultured cells used in the assay. This test method is based on established procedures for in vitro micronucleus testing.

[0161] N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was tested in all treatments up to the regulatory maximum dose level of 1 mM (260 mg / mL). There was no precipitation and only slight cytotoxicity at the 27 hour treatment. The relative increase in cell count (RICC) at 260 mg / mL compared to the negative control run simultaneously was 105% in the presence of S-9mix for 3 hour treatment, 98% in the absence of S-9mix for 3 hour treatment, and 74% in the absence of S-9mix for 27 hour treatment.

[0162] There was no statistically significant increase in MN percentage (%MN) with 3-hour treatment in the presence of S-9mix or with either 3-hour or 27-hour treatment in the absence of S-9mix. There was a significant trend with 3-hour treatment in the absence of S-9mix, but since there was no statistically significant increase and all %MN counts were within the 95% control limits of the HCD, this was considered irrelevant.

[0163] Under the test conditions, N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was neither clastogenic nor aneugenic in the presence or absence of S9mix.

[0164] Embodiment 18: Inhibition of trypsin by secreted human Z A1AT in the presence of compounds of the invention in huZ mice

[0165] The purpose of this experiment was to purify human Z A1AT from three HuZ mice (22.1e, 24.1a, 24.1b) that had been treated with the compound N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide and then test the activity of A1AT by reacting with increasing amounts of trypsin. The reaction of A1AT with trypsin was analyzed by Western blot, probing with anti-human A1AT antibody. Control plasma from wild-type mice (15.5b) treated with the same compound was also included in the study.

[0166] method

[0167] Mice were treated with 200 mg / kg of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide by oral gavage twice daily for 10 consecutive days. Mice had free access to food (standard mouse chow, safe diet) and water. On study day 10, each mouse was dosed 3.5-4.5 hours before terminal procedures. Blood was collected from each mouse by cardiac puncture and collected into tubes containing EDTA 25, and plasma was prepared by centrifugation at 2700 x g for 10 minutes at 4°C. Aliquots of plasma were taken and stored at -80°C prior to analysis.

[0168] Prior to purification, 100 μl of mouse plasma was diluted to 300 μl with 20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.4 (reaction and wash buffer). Washed α1-antitrypsin selection beads (150 μl) (GE LifeSciences) were then added to the plasma and mixed on a rocker for 30 minutes. The beads were then separated from the plasma by centrifugation using a spin column (ThermoScientific). After washing the beads with 5 x 100 μl reaction buffer, A1AT was then eluted from the beads with 3 x 100 μl of 20 mM Tris, 150 mM NaCl, 0.6 M MgCl2, 5 mM EDTA, pH 7.4. A1AT was eluted directly into 300 ul reaction buffer to ensure that A1AT would not aggregate. Residual A1AT in plasma and plasma supernatant was quantified using human serpinA1 ELISA (human serine protease inhibitor A1 / α1-antitrypsin dual recombinant ELISA, R&D Systems, DY1268) to confirm the consumption of human A1AT. The concentration of the eluted fractions was also measured. The amount of eluted A1AT was determined by A280 measurement using an extinction coefficient of 0.45.

[0169] The activity of the eluted A1AT was tested by evaluating its ability to form a complex with trypsin. The reaction mixture was prepared as shown in Table 4. The reaction was incubated for 5 minutes, and then 1 μl of 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride was added. After another 10 minutes, 21 μl of SDS sample buffer was added and boiled for 5 minutes. The samples were electrophoresed on a 10% SDS gel and blotted onto a nitrocellulose membrane. The membrane was then blocked with PBS 0.1% triton x-100, 5% skim milk powder and then probed with rabbit anti-human α-1 anti-trypsin antibody (AbD serotec 0640-5039) at 4°C overnight. After washing the membrane with PBS, 0.1% triton X-100, 0.1% skim milk, the blot was probed with anti-rabbit IgG HRP conjugate. After further washing the membrane, the blot was developed using SuperSignal West pico PLUS chemiluminescent substrate (Thermo Scientific). The results are shown in Figure 4. Fig. 7A and 7B shown.

[0170] Table 4. Experimental conditions for various treatments of isolated Z A1AT

[0171]

[0172] result

[0173] Fig. 7A and 7B The data in the literature show that in wild-type control mice (15.5b – see Figure 7B Human A1AT was not detected in plasma from huZ mice. Human Z A1AT was detected in the other three samples from huZ mice (see Fig. 7A and 7B Upon reaction with increasing amounts of trypsin, all samples exhibited complete reaction with trypsin, as indicated by the loss of the native band (labeled "NA") and the generation of a complex ("C") or cleaved band ("CA"), indicating that the Z A1AT in the eluted fractions was native and fully active.

Claims

1. A compound, which is N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

2. A compound, which is N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

3. A compound, which is N,N-bis(methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

4. A pharmaceutically acceptable salt form of a compound according to any one of claims 1 to 3.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt form according to claim 4 and a pharmaceutically or therapeutically acceptable excipient or carrier.

6. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt form according to claim 4 for the preparation of a medicament for the treatment of alpha 1 -antitrypsin deficiency (AATD).

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