Pyrrolidine compounds lp(a) lowering activity

Abstract

The present disclosure provides compounds of the formula: and their pharmaceutically acceptable salts, as well as pharmaceutical compositions comprising these compounds and their use in the treatment of cardiovascular disease and elevated Lp(a) plasma levels.

Description

Pyrrolidine Compounds with Lp(a) Lowering Activity FIELD OF THE INVENTION

[0001] This invention relates to pyrrolidine compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions, and therapeutic uses of the compounds, in particular their use in lowering lipoprotein(a) (Lp(a)) plasma levels. BACKGROUND OF THE INVENTION

[0002] There have been significant advances in treating cardiovascular disease (CVD). Despite treatment advances, patients continue to experience cardiovascular disease events such as angina, myocardial infarction, and stroke, which if untreated, lead to death. Lipid disorder or dyslipidemia remains a major risk factor for CVD. Lipid disorders can be divided into four general risk factors: elevated low-density lipoprotein cholesterol (LDL-c), low high-density lipoprotein cholesterol (HDL-c), elevated triglycerides (TG), and elevated Lp(a). There are a variety of treatment regimens targeting elevated LDL-c, low HDL-c, and elevated triglycerides. There are few approved treatment options for patients with elevated Lp(a) concentrations. In some cases, apheresis may be used to filter the blood to remove LDL and Lp(a); however, the effects are temporary and typically need to be repeated every two weeks. There is currently no pharmaceutical treatment approved specifically to lower Lp(a) levels.

[0003] Lp(a) is a genetically determined, independent cardiovascular risk factor. Elevated serum Lp(a) levels greater than 50 mg / dL or 125 nmol / L found in ~20% of the population confer at least a 1.6-fold risk of a first cardiovascular event (Kronenberg, F. Clin. Res. Cardiol. Suppl. 14, 5-12 (2019)) and a >1.42-fold increase risk of a second event (Madsen, C. M. et al. Arterioscler. Thromb. Vasc. Biol.40, 255-266 (2020)). Lp(a) may exhibit both prothrombotic and antithrombotic properties, and atherogenic and atherothrombotic properties. Lp(a) may inhibit fibrinolysis and accumulate in the vascular wall inducing thrombogenesis and atherosclerotic lesions. Plasma levels of Lp(a) vary substantially among individuals. Unlike the other risk factors, Lp(a) plasma levels do not vary significantly with diet and exercise.

[0004] Lp(a) resembles LDL-c in that it includes an LDL lipid core with the attendant apolipoprotein B (apoB), but unlike LDL-c, Lp(a) also contains a unique apolipoprotein(a) (apo(a)) bound to the apoB via disulfide bond. Apo(a) is synthesized in the liver. The assemblyof Lp(a) from apo(a) and LDL particles can occur in hepatocytes, on the cell wall, or in plasma. Inhibition of the assembly of the LDL particle with apo(a) reduces Lp(a) levels.

[0005] WO2020 / 247429 discloses compounds which inhibit the formation of Lp(a) by blocking the interaction between Apo(a) and ApoB.

[0006] Additional treatment options are desired for patients suffering from cardiovascular diseases and, in particular, patients suffering from lipid disorders or dyslipidemia. There is a need for additional treatment options for patients whose cardiovascular risks are not adequately managed using current standard of care therapies, such as, diet, exercise and / or the use of one or more drugs such as statins, fibrates, and niacin. More particularly, there is a need for further, alternative pharmaceutically acceptable compounds which inhibit the formation of Lp(a) and thus, reduce plasma Lp(a) levels. SUMMARY OF THE INVENTION

[0007] Accordingly, in a first aspect, there is provided a compound of Formula I:Z at each occurrence is independently H, C1-4alkyl, OH or cyclopropyl; and Y at each occurrence is independently CH2, O or S, or a pharmaceutically acceptable salt thereof.

[0008] In a second aspect, there is provided a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0009] In a third aspect, there is provided a method of treating cardiovascular disease in a patient comprising administering to the patient an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

[0010] In a fourth aspect, there is provided a method of treating elevated Lp(a) plasma levels in a patient comprising administering to the patient an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

[0011] In a fifth aspect, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0012] In a sixth aspect, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of cardiovascular disease.

[0013] In a seventh aspect, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of elevated Lp(a) plasma levels. DETAILED DESCRIPTION OF THE INVENTION

[0014] In an embodiment of a compound of Formula I, Z at each occurrence is H.

[0015] In an embodiment of a compound of Formula I, Y at each occurrence is CH2.

[0016] In an embodiment, there is provided a compound of Formula II:, II or a pharmaceutically acceptable salt thereof. Formula I encompasses Formula II and reference to Formula I below, for example in the methods of treatment and therapeutic uses, is also to be read as a reference to Formula II.

[0017] In an embodiment, the compound of Formula I is selected from:or a pharmaceutically acceptable salt thereof.

[0018] In an embodiment, there is provided a method of treating a patient in need of treatment for cardiovascular disease, comprising administering an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In an embodiment, there is provided a method of treating a patient in need of treatment for elevated Lp(a) plasma levels, comprising administering an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

[0019] In an embodiment, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0020] In an embodiment, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of cardiovascular disease. In an embodiment, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in treating elevated Lp(a) plasma levels.

[0021] In an embodiment, there is provided the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cardiovascular disease. In an embodiment, there is provided the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of elevated Lp(a) plasma levels.

[0022] The term “C1-4 alkyl” refers to a straight or branched chain saturated hydrocarbon containing 1 to 4 carbon atoms. Examples of a C1-4 alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, and tert-butyl.

[0023] As used herein, the term “elevated Lp(a) plasma levels” means a plasma level of Lp(a) that is equal to or above about 50 mg / dL. A compound provided herein may be used in treatment to reduce Lp(a) plasma levels.

[0024] The term “pharmaceutically acceptable salt” as used herein refers a salt of a compound that is acceptable for clinical and / or veterinary use. Examples of pharmaceutically acceptable salts and common methodology for preparing them can be found in “Handbook of Pharmaceutical Salts: Properties, Selection and Use” P. Stahl, et al., 2nd Revised Edition, Wiley- VCH, 2011 and S.M. Berge, et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19. In particular, the compounds of Formula I may be a zwitterion, a mono-, di, or tri-acid addition salt.

[0025] The pharmaceutical compositions for the present invention may be prepared using pharmaceutically acceptable additives. The term “pharmaceutically acceptable” refers to one or more carriers, diluents, and / or excipients that are compatible with the other components of the composition and not pharmaceutically deleterious to the patient. Examples of pharmaceutical compositions and processes for their preparation are well known to the skilled artisan, and can be found, for example, in “Remington: The Science and Practice of Pharmacy”, Loyd, V., et al. Eds., 22nd Ed., Mack Publishing Co., 2012.

[0026] As used herein, the term “effective amount” refers to a dosage amount that is effective in treating a disorder. The effective amount for a particular patient can be determined by a skilled health professional.

[0027] As used herein, the terms “treating”, “to treat”, or “treatment”, includes slowing, reducing, preventing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. As used herein, “treating cardiovascular disease” means slowing, reducing, preventing, or reversing the progression of heart or blood vessel disease.

[0028] As used herein, the term "patient" refers to a mammal. Preferably, the patient is a human.

[0029] Pharmaceutical compositions can be formulated as a tablet or capsule for oral administration, a solution for oral administration, or an injectable solution. In an embodiment the composition is suitable for oral administration.

[0030] The present invention contemplates all individual enantiomers, diastereomers, mixtures thereof, and racemates, of compounds of Formula I, and pharmaceutically acceptable salts thereof.

[0031] Certain abbreviations may refer to the following: “ACN” refers to acetonitrile; “Apo” refers to Apolipoprotein; “BOC” refers to tert-butoxycarbonyl; “DCM” refers to dichloromethane; “DMA” refers dimethylacetamide; “DMEM” refers to Dulbecco’s Modified Eagle’s Medium; “DMSO” refers to dimethyl sulfoxide; “EACA” refers to epsilon-aminocaproic acid or 6-aminocaproic acid; “ELISA” refers to enzyme-linked immunosorbent assay; “EtOAc” refers to ethyl acetate; “FBS” refers to Fetal Bovine Serum; “HEC” refers to hydroxy ethyl cellulose; “HEK” refers to human embryonic kidney; “HepG2” refers to a human hepatoma cell line; “HEPES” refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; “HLB” refers to hydrophilic-lipophilic balance; “hr / s” refers to hour / hours; “HRP” refers to Horseradish Peroxidase; “IC50” refers to the concentration of an agent that produces 50% of the maximal inhibitory response possible for that agent; “MeCN” refers to acetonitrile; “min” refers to minute / s; “MTBE” refers to methyl tert-butyl ether; “RT” refers to room temperature; “TFA” refers to trifluoroacetic acid; “THF” refers to tetrahydrofuran; and “TMB” refers to 3,3’,5,5’- teramethylbenzidine.

[0032] Individual isomers, enantiomers, and diastereomers may be separated or resolved by one of ordinary skill in the art at any convenient point in the synthesis of compounds listed below, by methods known to the artisan, such as selective crystallization techniques or chiral chromatography.

[0033] A compound of Formula I is readily converted to and may be isolated as a pharmaceutically acceptable salt. Salt formation can occur upon the addition of a pharmaceutically acceptable acid to form the acid addition salt or by the addition of a pharmaceutically acceptable base to form a base addition salt. Salts can also form simultaneously upon deprotection of a nitrogen or oxygen, i.e., removing the protecting group. Examples, reactions and conditions for salt formation are known to the skilled artisan.

[0034] The compounds of Formula I or any depicted formulae, or salts thereof, may be prepared by a variety of procedures, some of which are illustrated in the Preparations and Examples below. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different routes, to prepare compounds or salts of the present invention. The products of each step in the Preparations below can be recovered by conventional methods, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization.

[0035] In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. Without limiting the scope of the invention, the following schemes, preparations, and examples are provided to further illustrate the invention. Compounds of afore-depicted formulae, or salts thereof may be prepared by using starting materials or intermediates with the corresponding desired stereochemical configuration. Scheme 1

[0036] In Scheme 1, Step A depicts conversion of compound (1) to compound (3). Compound (1) is reacted with a bromobenzene compound (2), such as 1,3,5- tris(bromomethyl)benzene, 1,3-bis(bromomethyl)benzene or 1,4-bis(bromomethyl)benzene in the presence of lithium bis(trimethylsilyl)amide as a base and a solvent such as THF to give compound (3).

[0037] Step B shows a hydrolysis reaction in which oxazolidinone in compound (3) is cleaved by using LiOH / H2O2 in the presence of a solvent such as THF to give compound (4).

[0038] Step C shows acid deprotection of compound (4) in which BOC protecting groups are removed in the presence of HCl to the give amine as an HCl salt [compound (5) – a compound of Formula I].

[0039] In Scheme 2, Step A depicts conversion of compound (6) to compound (7). Compound (6) is reacted with 4-fluoro-3-nitrobenzyl bromide in the presence of lithium bis(trimethylsilyl)amide as a base and a solvent such as THF to give compound (7).

[0040] Step B shows a hydrolysis reaction in which oxazolidinone in compound (7) is cleaved by using LiOH / H2O2 in the presence of a solvent such as THF to give compound (8).

[0041] Step C depicts formation of a tert-butyl ester, compound (9) by reacting compound (8) with 2-tert-butyl-1,3-diisopropylisourea in the presence of a solvent such as 2- methyltetrahydrofuran.

[0042] Step D shows a reaction of 2-phenylethanamine with compound (9) in the presence of DMA as a solvent, followed by the addition of tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl- 3-oxo-propyl]pyrrolidine-1-carboxylate to give compound (10).

[0043] Step E a deprotection process in which the BOC groups in compound (10) are removed under acidic conditions (e.g. HCl), to give compound (11) (a compound of Formula I). Preparation 1 Di-tert-butyl 3,3'-((2S,2'S)-1,3-phenylenebis(3-((S)-4-benzyl-2-oxooxazolidin-3-yl)-3- oxopropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0044] Lithium bis(trimethylsilyl)amide (1 M in THF, 4.6 mL) was added to a solution of tert-butyl (3R)-3-[2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-2-oxo-ethyl]pyrrolidine-1- carboxylate (prepared essentially as described in WO 2020 / 247429, 1.7 g, 4.4 mmol) in THF (12.9 mL) at 0 ºC. The mixture was stirred for 30 min at 0 °C. A solution of 1,3- bis(bromomethyl)benzene (0.5 g, 1.8 mmol) was added dropwise in THF (5.5 mL), and the resulting mixture allowed to reach RT and stirred overnight. Saturated NH4Cl was added and extracted with EtOAc. The organic layer was washed with H2O and diatomaceous earth. The organic layer was dried over MgSO4, and the solution concentrated under vacuum, to give the title compound (1.5 g, 93%) as a dark yellow oil. ES / MS (m / z): 780 [M+H-BOC].Preparation 2 (2S,2'S)-3,3'-(1,3-Phenylene)bis(2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoic acid)

[0045] A mixture of hydrogen peroxide (6.9 mL, 68 mmol) and lithium hydroxide (0.8 g, 34 mmol) was added to a solution of di-tert-butyl 3,3'-((2S,2'S)-1,3-phenylenebis(3-((S)-4-benzyl-2- oxooxazolidin-3-yl)-3-oxopropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate) (1.5 g, 1.7 mmol) in THF (17 mL) at 0 °C. The mixture was stirred for 5 min at 0 °C, and then at RT overnight. NaHSO340% in water was added at 0 ºC to quench the reaction and allowed to reach RT. pH was adjusted to about 3 with 1N HCl in water. The mixture was extracted with EtOAc. The organics were washed with saturated aqueous NaC1, dried over MgSO4, filtered, and evaporated to dryness. The resulting residue was purified by reverse phase chromatography: Column: XBridge C18TM(19 x 100 mm, 5µm); Mobile Phase: 20mM ammonium bicarbonate in water-pH9 (A) / MeCN (B); Elution conditions: gradient 25% of ammonium bicarbonate over 6 mins; Flow Rate: 25mL / min; RT; Collection was guided by MS (ESI + mode), to give the title compound (90 mg, 9%). ES / MS (m / z): 562 [M+H].Preparation 3 Tri-tert-butyl 3,3',3''-((2S,2'S,2''S)-benzene-1,3,5-triyltris(3-((S)-4-benzyl-2-oxooxazolidin-3-yl)- 3-oxopropane-1,2-diyl))(3R,3'R,3''R)-tris(pyrrolidine-1-carboxylate)

[0046] Lithium bis(trimethylsilyl)amide (1 M in THF, 5.7 mL, 5.7 mmol) was added to a solution of tert-butyl (3R)-3-[2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-2-oxo-ethyl]pyrrolidine-1- carboxylate (prepared essentially as described in WO 2020 / 247429, 2 g, 5.22 mmol) in THF (12 mL) and cooled at 0 °C. The mixture was stirred for 30 min and then added a solution of 1,3,5- tris(bromomethyl)benzene (600 mg, 1.63 mmol) in THF (5 mL). The mixture was allowed to warm up to RT and stirred overnight. Saturated NH4Cl was added and diluted the mixture with EtOAc. The phases were separated and dried the organic phase over MgSO4, filtered, and concentrated the filtrate under vacuum to give the title compound, which was carried forward into Preparation 4 without further characterization or purification. Preparation 4 (2S,2'S,2''S)-3,3',3''-(Benzene-1,3,5-triyl)tris(2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3- yl)propanoic acid)

[0047] The title compound was prepared essentially as described in Preparation 2 using tri- tert-butyl 3,3',3''-((2S,2'S,2''S)-benzene-1,3,5-triyltris(3-((S)-4-benzyl-2-oxooxazolidin-3-yl)-3- oxopropane-1,2-diyl))(3R,3'R,3''R)-tris(pyrrolidine-1-carboxylate. ES / MS m / z: 702 [M+H-Boc]. Preparation 5 tert-Butyl (3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-1-[[4-[(2S)-3-[(4S)-4-benzyl-2- oxo-oxazolidin-3-yl]-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxo- propyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate

[0048] The title compound was prepared essentially as described in Preparation 1 using 1,4- bis(bromomethyl)benzene, and purified by silica gel chromatography using a gradient of 0 to 60% EtOAc in hexanes. ES / MS 779 [M+H-Boc].Preparation 6 tert-Butyl (3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-1-[(4-fluoro-3-nitro- phenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate

[0049] Lithium bis(trimethylsilyl)amide (1 M) in THF (14 mL, 14 mmol) was added to a solution of tert-butyl (3R)-3-[2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-2-oxo-ethyl]pyrrolidine-1- carboxylate (prepared essentially as described in WO 2020 / 247429, 4.5 g, 12 mmol) in THF (81 mL) at 0 ºC. The mixture was stirred for 30 min. A solution of 4-fluoro-3-nitrobenzyl bromide (3.0 g, 13 mmol) in THF (14 mL) was added dropwise and the resulting mixture allowed to reach RT. The mixture was quenched with saturated NH4Cl and extracted with EtOAc. The combined organic phases were washed with saturated aqueous NaCl, dried over anhydrous MgSO4, filtered, and evaporated to dryness. The residue was purified using silica gel flash chromatography using a gradient of 8-40% acetone in hexanes to give the title compound (4.3 g, 48%) as a brown oil. ES / MS m / z: 486 [M-tBu+H]. Preparation 7 (2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-3-(4-fluoro-3-nitro-phenyl)propanoic acid

[0050] The title compound was prepared essentially as described in Preparation 2 using tert- butyl (3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-1-[(4-fluoro-3-nitro-phenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate, purified by silica gel chromatography using a gradient of 20-80% (1% acetic acid in EtOAc) in hexanes. ES / MS m / z 283 (M-BOC+H); 327 (M-tBu+H). Preparation 8 tert-Butyl (3R)-3-[(1S)-2-tert-butoxy-1-[(4-fluoro-3-nitro-phenyl)methyl]-2-oxo- ethyl]pyrrolidine-1-carboxylate

[0051] To a suspension of (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-(4-fluoro-3- nitro-phenyl)propanoic acid (1.078 g, 2.819 mmol) in 2-methyltetrahydrofuran (17 mL) was added 2-tert-butyl-1,3-diisopropylisourea (1.37 g, 6.48 mmol) and the mixture was heated at 55 ºC for 30 min. Additional 2-tert-butyl-1,3-diisopropylisourea (1.37 g, 6.48 mmol) was added and the mixture was heated at 55 ºC for 40 min. After 40 min, additional 2-tert-butyl-1,3- diisopropylisourea (0.8917 g, 4.229 mmol) was added and the mixture was heated at 55ºC for 1 hr. The reaction mixture was allowed to reach RT and MTBE was added, the white solid filtered, washed with MTBE and discarded. The filtrate was evaporated to dryness. The residue was purified by silica gel flash column chromatography eluting with hexane in acetone (7 to 30% gradient) to give the title compound (1 g, 81%) as a pale-yellow oil. ES / MS 461 [M+23]. Preparation 9 tert-Butyl (3R)-3-[(1S)-1-[(4S)-4-benzyl-2-oxo-oxazolidine-3-carbonyl]but-3-enyl]pyrrolidine-1- carboxylate

[0052] The title compound was prepared essentially as described in Preparation 6 using allyl bromide, purified by silica gel chromatography using a gradient of 0-100% acetone in hexanes. ES-MS m / z 373 (M-tBu+H). Preparation 10 (2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]pent-4-enoic acid

[0053] The title compound was prepared essentially as described in Preparation 7 using tert- butyl (3R)-3-[(1S)-1-[(4S)-4-benzyl-2-oxo-oxazolidine-3-carbonyl]but-3-enyl]pyrrolidine-1- carboxylate, purified by silica gel chromatography using a gradient of 20-80% EtOAc in (hexanes + 1% acetic acid). ES-MS m / z 214 (M-tBu+H). Preparation 11 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonylbut-3-enyl]pyrrolidine-1-carboxylate

[0054] The title compound was prepared essentially as described in Preparation 8 using (2S)- 2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]pent-4-enoic acid, purified by silica gel chromatography using a gradient of 0-30% acetone in hexanes. ES-MS m / z 214(M- 2xtBu+H). Preparation 12 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-3-oxo-propyl]pyrrolidine-1-carboxylate

[0055] To a mixture of tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonylbut-3-enyl]pyrrolidine- 1-carboxylate (260 mg, 0.7990 mmol) in THF (7.7 mL) and water (3.2 mL) at 0ºC was addedsodium periodate (0.68 g, 3.20 mmol). Then, osmium tetroxide (4 mass% in water, 711 mg, 0.11 mmol) was added and the resulting mixture was stirred for 20 min at 0 °C, then at RT overnight. The mixture was quenched with water and extracted with MTBE. The combined organic phases were washed with brine, dried over MgSO4, filtered and evaporated to dryness. The residue was purified by silica gel chromatography (0-100% EtOAc in hexanes) to give 112 mg (43%) of the title compound as a colorless oil. ES-MS m / z 216 (M- 2xtBu+H). Preparation 13 Di-tert-butyl 3,3'-((2S,2'S)-(1-phenethyl-1H-benzo[d]imidazole-2,5-diyl)bis(3-(tert-butoxy)-3- oxopropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0056] 2-Phenylethanamine (18 µmol, 0.118 mmol, 100 mass%) was added to a mixture of tert-butyl (3R)-3-[(1S)-2-tert-butoxy-1-[(4-fluoro-3-nitro-phenyl)methyl]-2-oxo- ethyl]pyrrolidine-1-carboxylate (50 mg, 0.114 mmol) and DMA (980 µL). The reaction was incubated at 90 ºC for 2 hrs. Na2S2O4 (2M aqueous, 295 µL, 0.59 mmol) was added followed by addition of tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-3-oxo-propyl]pyrrolidine-1-carboxylate (118 µmol, 0.118 mmol). The reaction was stirred at RT for about 2 hrs., then at 90 °C, then purified on a HLB cartridge (6 g, 2 CV water, 2 CV ACN). The organic layer was separated, and the solvent was removed under N2 to give the title compound (28.5 mg, 31 %). The compound was used to prepare Example 4 without characterization. Example 1 (2S,2'S)-3,3'-(1,3-Phenylene)bis(2-((R)-pyrrolidin-3-yl)propanoic acid) dihydrochloride

[0057] HCl (2 M in diethyl ether, 2 mL, 4 mmol) was added to a solution of (2S,2'S)-3,3'- (1,3-phenylene)bis(2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoic acid) (90 mg, 0.16 mmol) in DCM (2.4 mL) and the mixture was stirred at RT overnight. The mixture was then concentrated to dryness under nitrogen stream at 40 ºC, triturated the solid with MTBE, and sonicated, filtered, washed with MTBE, and dried under high vacuum. The solid was dissolved in the minimum amount of water possible and concentrated to dryness under nitrogen stream at 40 ºC. The solid was dried in an oven at 45 ºC under high vacuum to give the title compound as pale-yellow solid (69 mg, 99%). ES / MS m / z: 361 [M+H]. Example 2 (2S,2'S,2''S)-3,3',3''-(Benzene-1,3,5-triyl)tris(2-((R)-pyrrolidin-3-yl)propanoic acid) trihydrochloride

[0058] The title compound was prepared essentially as described in Example 1 using (2S,2'S,2''S)-3,3',3''-(benzene-1,3,5-triyl)tris(2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3- yl)propanoic acid). ES / MS (m / z): 502 [M+H]. Example 3 (2S)-3-[4-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]-2-[(3R)-pyrrolidin-3- yl]propanoic acid;dihydrochloride

[0059] A mixture of hydrogen peroxide (30 mass % in water, 2.04 mL, 18 mmol) and lithium hydroxide (218 mg, 9.1 mmol) was added to solution of tert-butyl (3R)-3-[(1S)-2-[(4S)- 4-benzyl-2-oxo-oxazolidin-3-yl]-1-[[4-[(2S)-3-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-2-[(3R)-1- tert-butoxycarbonylpyrrolidin-3-yl]-3-oxo-propyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1- carboxylate (2.0 g, 2.3 mmol) in THF ( 23 mL) at 0 °C . The reaction was stirred at RT for 2 hrs. Reaction was quenched with NaOH (1 N) and crude product was washed with EtOAc. The aqueous phase was acidified with HCl (1 N) and extracted with EtOAc then DCM. The combined organic extraction phases were dried over MgSO4 and evaporated under reduced pressure. The residue was purified with silica gel chromatography eluting with hexane in acetone (10-80 %) giving a clear oil which foamed under vacuum. The foam was dissolved in DCM and added HCl (4 M in 1,4-dioxane, 1 mL, 4 mmol). The reaction was sealed and stirred at RT overnight, then concentrated to give a white solid. The white solid was purified by reverse phase chromatography [column: 30 × 100 mm, 5 µm; flow rate: 85 mL / min; mobile phase: 20% MeCN:0.4% (v / v) TFA in water isocratic] to give the title compound as a TFA salt. The TFA salt was diluted in 5N aq. HCl and concentrated under reduced pressure. The resulting oil was then suspended in HCl (4 M in 1,4-dioxane). This white suspension was filtered and the solid dried overnight in a vacuum oven to give the title compound (192 mg, 19%) as an HCl salt. ES / MS m / z: 361 [M+H]. Example 4 (2S,2'S)-3,3'-(1-phenethyl-1H-benzo[d]imidazole-2,5-diyl)bis(2-((R)-pyrrolidin-3-yl)propanoic acid) trihydrochloride

[0060] The title compound was prepared essentially as described in Example 1 using di-tert- butyl 3,3'-((2S,2'S)-(1-phenethyl-1H-benzo[d]imidazole-2,5-diyl)bis(3-(tert-butoxy)-3- oxopropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate), purified on a HLB cartridge (eluted with water and ACN). ES / MS m / z 505 [M+H]. In vitro Lp(a) Assembly Assay

[0061] The ability of compounds to inhibit the formation of Lp(a) particles in vitro was assessed by a cell-free assembly assay. Conditioned media (DMEM supplemented with 10% FBS, 20 mM HEPES, and 1x penicillin / streptomycin) was collected from confluent wild-type HepG2 cells (a source of endogenously expressed ApoB) and from a HEK293 stable cell line expressing human Apo(a) protein containing 17 Kringle repeats (selected on 1 mg / ml geneticin) after 24 h

[0062]

[0063] of culture at 37 °C and 5% CO2. An in vitro assembly assay was conducted by combining equal parts of HepG2 and HEK293 conditioned media with the test compounds added in dilution series (final concentration 0.01~100 nM). The reaction was incubated at 37 °C for 2 hrs and then stopped with the addition of EACA to a final concentration of 150 mM. Lp(a) was detected using a sandwich ELISA with an anti-Lp(a) capture antibody and an HRP-conjugated anti-ApoB detection antibody. The ELISA was developed using TMB, stopped using 1 N sulfuric acid, and the signal was read at 450 nm on a Molecular Devices plate reader. The % inhibition of Lp(a) formed for each test condition was determined with an assembly reaction having no inhibitor present (with matched DMSO concentration at 1%) set to 0% inhibition, and an assembly reaction with a minimal amount of the HepG2 conditioned media present (50-fold dilution) set to 100% inhibition. Data were fitted to a 4-parameter curve to determine the IC50values summarized in Table 1. Addition of the Example test compound to conditioned media containing ApoB and Apo(a) lead to concentration-dependent inhibition of Lp(a) formation in vitro, as summarized in Table 1. The results indicate that these compounds inhibit the assembly of Lp(a) from Apo(a) and the LDL particle. Table 1

Claims

CLAIMS We claim:

1. A compound of the formula:Z at each occurrence is independently H, C1-4 alkyl, OH or cyclopropyl; and Y at each occurrence is independently CH2, O or S, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein Z at each occurrence is H, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 or claim 2, wherein Y at each occurrence is CH2, or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1, which is of the formula:whereinor a pharmaceutically acceptable salt thereof.

5. The compound according to claim 1, wherein the compound is selected from:or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 5 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

7. A method of treating cardiovascular disease in a patient comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof.

8. A method of treating elevated Lp(a) plasma levels in a patient comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof.

9. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for use in therapy.

10. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for use in the treatment of cardiovascular disease.

11. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for use in the treatment of elevated Lp(a) plasma levels.

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